**A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses**

Diogo Ribeiro Demartini

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54484

### **1. Introduction**

Mass spectrometry has been widely used for analyses of biomolecules such as proteins. The soft ionization methods available nowadays, the faster and more accurate mass spectrome‐ ters, a diversity of protein databases resulting from large scale genome studies and the ad‐ vances in the bioinformatics field for optimized data mining, altogether significantly contributed to high quality outputs in the proteomics area [1-3].

In order to achieve the higher number of identified proteins (and perhaps quantified), in proteomics studies, some steps are equally important and required: 1) sample preparation; 2) sample pre-fractionation; 3) peptidase digestion; 4) mass spectrometry analysis; 5) data mining. Each of these steps can be extremely challenging and the final conclusions will be based on their success [4-6].

Samples used for proteomics studies are always complex and the abundant proteins mask the low abundance proteins in several cases. Tryptic peptides originated from abundant proteins predominate, no matter what kind of analysis is performed in the mass spectrometer. Highly abundant proteins such as albumin in blood samples, cytoskeleton proteins in pelleted cells, among other examples, need to be removed to allow detection of less abundant ones, and re‐ quire separate analyses [7]. The same situation happens in plant derived materials, in which the amount of RuBisCO in leaves and of storage proteins in seeds and seedlings mask the non abun‐ dant proteins in the samples. To deal with this situation, several approaches can be used. For ex‐ ample, these proteins can be depleted from the source sample by immunoprecipitation. Another strategy used is to create exclusion lists for the mass spectrometer during data analy‐ sis. In this case, the selected peptides will be ignored during data acquisition. Another approach widely used is the dynamic exclusion. In this case, according to user definitions, the mass spec‐ trometer will ignore the most abundant peaks in the MS1 (for example, 5), and will analyze the

© 2013 Demartini; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Demartini; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

next top 5. However, if the mass spectrometer used is not fast enough during each cycle, impor‐ tant information could be lost [8,9].

**2.1. Ionization**

referred as soft ionization methods [13,16-18].

are detectable in most mass analyzers [13].

the liquid to the counter electrode [13,14].

decrease considerably in size and charges states [13,14].

*2.1.1. Electrospray Ionization (ESI)*

In order to be analyzed in a mass spectrometer, the sample must be ionized and it is impera‐ tive that the ionized molecules (proteins or peptides) turn into a gas phase to allow analysis, fragmentation and detection [14]. The extensive advance of mass spectrometry in protein analyses happened after the advance of the ESI (electrospray ionization) [16], and MALDI (matrix-assisted laser desorption/ionization) ionization methods, which allowed analysis by mass spectrometry to be extended to non-volatile and thermolabile compounds. In both cas‐ es, the production of intact molecular ions is achieved under adequate experimental condi‐ tions, with minimal fragmentation. Due to this fact, these two ionization processes are

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

41

Shortly, in electrospray ionization the sample (proteins or peptides) is nebulized when high voltage is applied. As previously said, this ionization process allowed extensive studies on proteomics field because almost no energy is retained by the analyte and, in general, no fragmentation happens during the ionization process [17,18]. Another important aspect is that it generates multiple charges species (specially for ionized peptides), and the *m/z* values

The ionization process is based on a liquid dispersion [14] and the process takes place fol‐ lowing three main steps: production of charged droplets, the fission of the charged droplets and production of desolvated ions [13,14,19,20]. The production of charged droplets takes place when high-voltage is applied at the capillary tip where the analyte solution is being injected. At this stage, the electric field causes a separation of the positive and negative charges in the solution containing the analyte. In case of operation in positive ion mode (when the capillary is set at positive potential), the positive ions move towards the counter electrode. It causes an accumulation of positive ions at the surface of the liquid in the tip. The reverse polarity will produce negatively charges. There is a deformation of the meniscus of the liquid in the tip at a critical potential forming the Taylor cone [16,17] which is a static description and does not include spraying behavior [14]. The electric potential applied to the liquid at the tip pulls it into an elliptic shape. However, there is an equilibrium between the surface tension trying to pull the liquid back and the electrostatic attraction which pushes

The capillary tip is under a constant neutral gas flow, such as nitrogen. In this case, the colli‐ sion of the gas with the droplet from the tip causes the solvent evaporation, a key step in the ESI method. The second step in the electrospray process is the Rayleigh fission of the drop‐ lets. The droplets fission happens when the Coulombic repulsion between the charges is stronger than the surface tension of the liquid, due to a constant decrease of the droplet radi‐ us. The limit in which this phenomenon happens is called the Rayleigh limit: the balance be‐ tween the surface tension and electrostatic attraction is lost. At this stage, the droplets

A nice example of a proteomic study is the quantification of 1,323 proteins from *Arabidopsis thaliana* chloroplasts*,* using label-free spectral counting. This was achieved by coupling or‐ ganelle fractionation (thylakoids and stroma) and different extractions methods, applied to enrich the abundant proteins fraction (ammonium acetate precipitation and alkaline extrac‐ tion), and analysis of all fraction separately. Protein pre-fractionation using sodium dodecyl sulfate one-dimension polyacrylamide gel electrophoresis and high-resolution mass spec‐ trometry analysis performed with two different machines, also contributed to this high number of identified and quantified proteins [10].

The challenge grows even more when the samples to be analyzed consist of membrane pro‐ teins [11]. Membrane proteins usually are highly hydrophobic and in this case chaotropic agents combined with high amounts of detergents are employed to make them soluble. However, these additives can interfere in the trypsin digestion process. The concentration of these agents must be reduced in order to maintain the trypsin activity. In case of *in solution* digestion, besides chaotropes and detergents, reducing and alkylation agents (usually di‐ thiothreitol and iodoacetimide, respectively) must also be removed. These steps, designated as desalting process, can be performed in several ways. Most commonly it involves reverse phase chromatography in which the pH of the digested sample (peptides) is reduced using trifluoroacetic acid, formic acid, or others. The peptides are bound to a C-18 matrix and elut‐ ed with increasing concentrations of acetonitrile. After drying the samples under vacuum, they are prepared for mass spectrometry analyses [3,4,12].

The mass spectrometer itself must be correctly chosen to obtain a satisfactory balance be‐ tween accuracy and speed. Fast scan rates instruments can lose accuracy in their measure‐ ments, and *vice versa.*

Tandem mass spectrometry (MS/MS) is a method where the gaseous ions are subjected to two or more sequential stages of mass analysis, which can be separated spatially or tempo‐ rally, according to their mass to charge ratio, *m/z* [13,14]. The mass analyzers characterize the ions according to their *m/z*. The ion selected in the first stage of analysis is subjected to different reactions and charged products from this reaction are analyzed in a second step (a second mass analyzer or other types of analyzers) [15]. The "reaction" step is critical for data quality and performance.

A brief overview of the most used components in a mass spectrometer applied in proteo‐ mics research is discussed, specially focused on the newest researchers on the field.

### **2. Most used components for mass spectrometers in proteomics analyses**

In this section a brief overview of the components of a mass spectrometer used in proteo‐ mics will be given.

### **2.1. Ionization**

next top 5. However, if the mass spectrometer used is not fast enough during each cycle, impor‐

A nice example of a proteomic study is the quantification of 1,323 proteins from *Arabidopsis thaliana* chloroplasts*,* using label-free spectral counting. This was achieved by coupling or‐ ganelle fractionation (thylakoids and stroma) and different extractions methods, applied to enrich the abundant proteins fraction (ammonium acetate precipitation and alkaline extrac‐ tion), and analysis of all fraction separately. Protein pre-fractionation using sodium dodecyl sulfate one-dimension polyacrylamide gel electrophoresis and high-resolution mass spec‐ trometry analysis performed with two different machines, also contributed to this high

The challenge grows even more when the samples to be analyzed consist of membrane pro‐ teins [11]. Membrane proteins usually are highly hydrophobic and in this case chaotropic agents combined with high amounts of detergents are employed to make them soluble. However, these additives can interfere in the trypsin digestion process. The concentration of these agents must be reduced in order to maintain the trypsin activity. In case of *in solution* digestion, besides chaotropes and detergents, reducing and alkylation agents (usually di‐ thiothreitol and iodoacetimide, respectively) must also be removed. These steps, designated as desalting process, can be performed in several ways. Most commonly it involves reverse phase chromatography in which the pH of the digested sample (peptides) is reduced using trifluoroacetic acid, formic acid, or others. The peptides are bound to a C-18 matrix and elut‐ ed with increasing concentrations of acetonitrile. After drying the samples under vacuum,

The mass spectrometer itself must be correctly chosen to obtain a satisfactory balance be‐ tween accuracy and speed. Fast scan rates instruments can lose accuracy in their measure‐

Tandem mass spectrometry (MS/MS) is a method where the gaseous ions are subjected to two or more sequential stages of mass analysis, which can be separated spatially or tempo‐ rally, according to their mass to charge ratio, *m/z* [13,14]. The mass analyzers characterize the ions according to their *m/z*. The ion selected in the first stage of analysis is subjected to different reactions and charged products from this reaction are analyzed in a second step (a second mass analyzer or other types of analyzers) [15]. The "reaction" step is critical for data

A brief overview of the most used components in a mass spectrometer applied in proteo‐

**2. Most used components for mass spectrometers in proteomics analyses**

In this section a brief overview of the components of a mass spectrometer used in proteo‐

mics research is discussed, specially focused on the newest researchers on the field.

tant information could be lost [8,9].

40 Tandem Mass Spectrometry - Molecular Characterization

number of identified and quantified proteins [10].

they are prepared for mass spectrometry analyses [3,4,12].

ments, and *vice versa.*

quality and performance.

mics will be given.

In order to be analyzed in a mass spectrometer, the sample must be ionized and it is impera‐ tive that the ionized molecules (proteins or peptides) turn into a gas phase to allow analysis, fragmentation and detection [14]. The extensive advance of mass spectrometry in protein analyses happened after the advance of the ESI (electrospray ionization) [16], and MALDI (matrix-assisted laser desorption/ionization) ionization methods, which allowed analysis by mass spectrometry to be extended to non-volatile and thermolabile compounds. In both cas‐ es, the production of intact molecular ions is achieved under adequate experimental condi‐ tions, with minimal fragmentation. Due to this fact, these two ionization processes are referred as soft ionization methods [13,16-18].

### *2.1.1. Electrospray Ionization (ESI)*

Shortly, in electrospray ionization the sample (proteins or peptides) is nebulized when high voltage is applied. As previously said, this ionization process allowed extensive studies on proteomics field because almost no energy is retained by the analyte and, in general, no fragmentation happens during the ionization process [17,18]. Another important aspect is that it generates multiple charges species (specially for ionized peptides), and the *m/z* values are detectable in most mass analyzers [13].

The ionization process is based on a liquid dispersion [14] and the process takes place fol‐ lowing three main steps: production of charged droplets, the fission of the charged droplets and production of desolvated ions [13,14,19,20]. The production of charged droplets takes place when high-voltage is applied at the capillary tip where the analyte solution is being injected. At this stage, the electric field causes a separation of the positive and negative charges in the solution containing the analyte. In case of operation in positive ion mode (when the capillary is set at positive potential), the positive ions move towards the counter electrode. It causes an accumulation of positive ions at the surface of the liquid in the tip. The reverse polarity will produce negatively charges. There is a deformation of the meniscus of the liquid in the tip at a critical potential forming the Taylor cone [16,17] which is a static description and does not include spraying behavior [14]. The electric potential applied to the liquid at the tip pulls it into an elliptic shape. However, there is an equilibrium between the surface tension trying to pull the liquid back and the electrostatic attraction which pushes the liquid to the counter electrode [13,14].

The capillary tip is under a constant neutral gas flow, such as nitrogen. In this case, the colli‐ sion of the gas with the droplet from the tip causes the solvent evaporation, a key step in the ESI method. The second step in the electrospray process is the Rayleigh fission of the drop‐ lets. The droplets fission happens when the Coulombic repulsion between the charges is stronger than the surface tension of the liquid, due to a constant decrease of the droplet radi‐ us. The limit in which this phenomenon happens is called the Rayleigh limit: the balance be‐ tween the surface tension and electrostatic attraction is lost. At this stage, the droplets decrease considerably in size and charges states [13,14].

The next step is the solvent evaporation process from the charged droplet forming a gasphase ion-analyte [13]. Two main mechanisms explain the production of desolvated ions in the gas phase: ion evaporation mechanism and charged residue mechanism. The assump‐ tion of the ion evaporation mechanism is that, at a specific time, the electric field on the sur‐ face of the droplet is sufficiently high, which causes the emission of the solvated ion from the charged droplet [17,21]. The charged residue mechanism assumes that a series of fission events leads to a final droplet, which contains a single analyte molecule completely free of solvating solvent [17,22].

[25-28]. In case of proteins or peptides, the proton transfer mechanism is probably the most important secondary reaction. In most proteomics approaches, samples are spotted in a MALDI plate with acidic matrixes, and data collected in positive mode. Some analytes do not have a high proton affinity, then negative ions could be collected or the sample could be prepared with a more basic matrix [26]. Other important types of secondary reaction mecha‐ nisms that take place in the MALDI plume are the cation transfer, electron transfer and elec‐

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

43

Ionization by MALDI can be coupled to a liquid chromatography system similar to ESI, however, since the sample must be mixed with the matrix prior analysis, a spotter must be used. MALDI works with wide dynamic ranges [29], such as 2 kDa up to 7 kDa, or more [30]. The dominance of singly charged ions, specially for proteins or peptides with molecu‐ lar weighs 20 kDa is a characteristic of MALDI, in contrast with ESI which produces much

An important advantage of MALDI is that this ionization process is more tolerant to salts and high concentrations of buffer. However, determination of lower masses can be some‐ times difficult. This happens because the matrix is also ionized in the process and "flies" in the same range of low molecular masses molecules [32,33]. Most of the matrices used nowa‐ days are small organic molecules, which absorb UV in the range of 266– 355 nm. Nowadays, the number of choices for matrixes is quite small for proteomics analyses, derived from ben‐ zoic or cinnamic acid. It is important to point that not all matrixes are useful only for certain

Different MALDI matrices that can be used according to the type of analytes [24-26,34]. In

**b.** 2,5-dihydroxybenzoic acid: absorption 337-353 nm, and can be used for proteins, pepti‐

**d.** α-cyano-4-hydroxycinnamic acid: absorption 337-353 nm and mostly used for peptides

When the sample solution is mixed with the matrix, placed/spotted in a MALDI plate, and dried without vacuum, the distribution of the sample is the plate can be quite distorted. However, if vacuum is used to help drying the sample, a better chemical distribution can be achieved in each sample spot [37]. In most cases, the sample preparation is done by the "dried droplet" method, which is quite simple. The sample and the matrix are separately dissolved in a common solvent system (such as 0.1 % trifluoroacetic acid), and then mixed either before or on the MALDI plate. After that, the solvent evaporation will take place, helped or not by a gas flow. By the end, the sample will be co-crystallized with the analyte

**c.** sinapinic acid: absorption 337-353 nm and widely used for proteins and peptides;

**e.** 3-hydroxy-picolinic acid: absorption 337-353 and it is suitable for nucleic acids;

all types of analytes [25], causing different ways to prepare the sample.

**a.** nicotinic acid: absorption at 266 nm and used for proteins and peptides;

case of biological samples, some matrixes can be selected [24,35,36]:

des, carbohydrates and some synthetic polymers;

tron capture [26].

analyses;

and will be ready for analyses.

more species in higher charges states [31].

An important advantage of ESI is that it can be easily coupled with liquid chromatography systems, specially those working at nanoflow range. In a *bottom up* approach, in which the proteins are digested with a peptidase and the resulting peptides are separated in a reverse phase column, the electrospray ionization is widely used. Typical columns used to the sepa‐ rate the complex sample mixture of peptides are made of reverse phase materials (C-18, 3– 10 μm diameter) packed into fused silica capillaries (12–100 μm diameter) with sintered sili‐ ca particles or silicate-polymerized ceramics as frits [5]. The dead volume between the at the end of the column and the ionization region must be as short as possible to avoid peak broadening and mixture of the peptides which were just separated in the reverse phase col‐ umn. The ESI process is the same when the sample is continuously infused without previ‐ ous separation in the LC system. In these cases, the flow must be adjusted to higher values to compensate the lack of the packed resin.

A capillary column coupled to a LC system and the ESI process is represented in figure 1A.

### *2.1.2. Matrix-Assisted Laser Desorption/Ionization (MALDI)*

In MALDI analyses, the sample must be mixed with matrix and spotted in a stainless steel plate prior the analysis in the mass spectrometer. The sample is co-crystallized with the ma‐ trix, which has an essential function in MALDI. The co-crystallized sample is ionized by short laser pulses (Figure 1B). Subsequently, the ions are accelerated and the time that they spend to flight in a vacuum tube to reach the detector is measured in a TOF (time-of-flight) analyzer [23].

The matrix has to absorb the laser energy via electronic or vibrational excitation and it must also isolate the analyte molecules by diluting during preparation/crystallization preventing their aggregation. Finally, it must be able to perform the sample ionization [24]. The ioniza‐ tion method by MALDI can be divided, according to Zenobi and co-workers, into two main categories. In the "primary ionization", the first ions are generated from neutral molecules, mostly matrix-derived ions. In the "secondary" ionization the ions come mostly from the an‐ alyte samples, with few contamination from the matrix [24,25]. The disintegration of the condensed phase by the laser energy has to take place without excessive destructive heating of the embedded analyte molecules. The most straightforward explanation for ions forma‐ tion in MALDI, assumes that ions from primary ionization result from a laser excitation of an absorbing organic material by molecule multiphoton ionization, which leads to a matrix radical cation [25,26]. The secondary ion formation mechanism take place in the MALDI plume, which is a solid-to-gas phase transition state formed, shortly after the laser pulse [25-28]. In case of proteins or peptides, the proton transfer mechanism is probably the most important secondary reaction. In most proteomics approaches, samples are spotted in a MALDI plate with acidic matrixes, and data collected in positive mode. Some analytes do not have a high proton affinity, then negative ions could be collected or the sample could be prepared with a more basic matrix [26]. Other important types of secondary reaction mecha‐ nisms that take place in the MALDI plume are the cation transfer, electron transfer and elec‐ tron capture [26].

The next step is the solvent evaporation process from the charged droplet forming a gasphase ion-analyte [13]. Two main mechanisms explain the production of desolvated ions in the gas phase: ion evaporation mechanism and charged residue mechanism. The assump‐ tion of the ion evaporation mechanism is that, at a specific time, the electric field on the sur‐ face of the droplet is sufficiently high, which causes the emission of the solvated ion from the charged droplet [17,21]. The charged residue mechanism assumes that a series of fission events leads to a final droplet, which contains a single analyte molecule completely free of

An important advantage of ESI is that it can be easily coupled with liquid chromatography systems, specially those working at nanoflow range. In a *bottom up* approach, in which the proteins are digested with a peptidase and the resulting peptides are separated in a reverse phase column, the electrospray ionization is widely used. Typical columns used to the sepa‐ rate the complex sample mixture of peptides are made of reverse phase materials (C-18, 3– 10 μm diameter) packed into fused silica capillaries (12–100 μm diameter) with sintered sili‐ ca particles or silicate-polymerized ceramics as frits [5]. The dead volume between the at the end of the column and the ionization region must be as short as possible to avoid peak broadening and mixture of the peptides which were just separated in the reverse phase col‐ umn. The ESI process is the same when the sample is continuously infused without previ‐ ous separation in the LC system. In these cases, the flow must be adjusted to higher values

A capillary column coupled to a LC system and the ESI process is represented in figure 1A.

In MALDI analyses, the sample must be mixed with matrix and spotted in a stainless steel plate prior the analysis in the mass spectrometer. The sample is co-crystallized with the ma‐ trix, which has an essential function in MALDI. The co-crystallized sample is ionized by short laser pulses (Figure 1B). Subsequently, the ions are accelerated and the time that they spend to flight in a vacuum tube to reach the detector is measured in a TOF (time-of-flight)

The matrix has to absorb the laser energy via electronic or vibrational excitation and it must also isolate the analyte molecules by diluting during preparation/crystallization preventing their aggregation. Finally, it must be able to perform the sample ionization [24]. The ioniza‐ tion method by MALDI can be divided, according to Zenobi and co-workers, into two main categories. In the "primary ionization", the first ions are generated from neutral molecules, mostly matrix-derived ions. In the "secondary" ionization the ions come mostly from the an‐ alyte samples, with few contamination from the matrix [24,25]. The disintegration of the condensed phase by the laser energy has to take place without excessive destructive heating of the embedded analyte molecules. The most straightforward explanation for ions forma‐ tion in MALDI, assumes that ions from primary ionization result from a laser excitation of an absorbing organic material by molecule multiphoton ionization, which leads to a matrix radical cation [25,26]. The secondary ion formation mechanism take place in the MALDI plume, which is a solid-to-gas phase transition state formed, shortly after the laser pulse

solvating solvent [17,22].

42 Tandem Mass Spectrometry - Molecular Characterization

analyzer [23].

to compensate the lack of the packed resin.

*2.1.2. Matrix-Assisted Laser Desorption/Ionization (MALDI)*

Ionization by MALDI can be coupled to a liquid chromatography system similar to ESI, however, since the sample must be mixed with the matrix prior analysis, a spotter must be used. MALDI works with wide dynamic ranges [29], such as 2 kDa up to 7 kDa, or more [30]. The dominance of singly charged ions, specially for proteins or peptides with molecu‐ lar weighs 20 kDa is a characteristic of MALDI, in contrast with ESI which produces much more species in higher charges states [31].

An important advantage of MALDI is that this ionization process is more tolerant to salts and high concentrations of buffer. However, determination of lower masses can be some‐ times difficult. This happens because the matrix is also ionized in the process and "flies" in the same range of low molecular masses molecules [32,33]. Most of the matrices used nowa‐ days are small organic molecules, which absorb UV in the range of 266– 355 nm. Nowadays, the number of choices for matrixes is quite small for proteomics analyses, derived from ben‐ zoic or cinnamic acid. It is important to point that not all matrixes are useful only for certain all types of analytes [25], causing different ways to prepare the sample.

Different MALDI matrices that can be used according to the type of analytes [24-26,34]. In case of biological samples, some matrixes can be selected [24,35,36]:


When the sample solution is mixed with the matrix, placed/spotted in a MALDI plate, and dried without vacuum, the distribution of the sample is the plate can be quite distorted. However, if vacuum is used to help drying the sample, a better chemical distribution can be achieved in each sample spot [37]. In most cases, the sample preparation is done by the "dried droplet" method, which is quite simple. The sample and the matrix are separately dissolved in a common solvent system (such as 0.1 % trifluoroacetic acid), and then mixed either before or on the MALDI plate. After that, the solvent evaporation will take place, helped or not by a gas flow. By the end, the sample will be co-crystallized with the analyte and will be ready for analyses.

In case the ions are separated "in space" (time of flight, TOF; sector; quadrupoles), the tech‐ niques are called beam techniques, because the ions "travel" across the analyzer in a "pulsed beam" mode. In this case, the MS or MS/MS analyses are performed in separated events. The ion trap analyzers, such as quadruploe ion traps and orbitraps characterize ions based on the frequency of their motion in a defined space [15]. Thus MS and MS/MS events can be performed in the same analyzer, being separated by time and not by "space" [15,40]. The an‐ alyzers use magnetic or electric fields, or even combinations of both to select ions. To avoid undesired collisions with neutral gases during analyses, the operation is performed under

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

45

A quadrupole analyzer can work as a linear ion trap, in which ions are confined radially by a two-dimensional (2D) radio frequency field, and axially by stopping potentials applied to

A quadrupole mass analyzer is widely used as a "filter" prior fragmentation of the desired ions. Basically it consists of four roods assembled in two pairs, as shown in figure 2A. The first two opposite roods have the same applied voltage, which is different from that of the second two opposite roods establishing a two dimensional quadrupole field in the *x-y* plane (Figure 2A) [38]. The mass analysis depends on the radio frequency, and direct current vol‐ tages which are applied to the four roods [1]. Due to that reason, ions travelling in the quad‐ rupole during analysis will be, at the same time, attracted by one set of roods and repulsed by the second set of roods. Considering the ion population being injected in a quadrupole analyzer, a selection can be made according to their *m/z* ratio, making some ions to have a stable trajectory in the analyzer, while a considerable number of other ions will not go all the way through (Figure 2A, arrows). The ion path occurs in the *z* direction, while the attraction and repulsion are occurring simultaneously in the *x* and *y* direction (Figure 2A) [38,42]. If the oscillations of an ion are stable, the ion will continue to drift down the rod assembly and reach the detector. The stable ions which "travel" all the analyzer length will go to the next

steps, which can be detection, fragmentation and a second round of analysis.

prior fragmentation in the collision cell, in the case of MS/MS analysis.

*2.2.2. Quadrupole ion trap analyzers*

The above explanation is quite simple for a complex situation. In a quadrupole mass filter ions of a single *m/z* maintain stable trajectories from the ion source to the detector, whereas ions with different *m/z* values are unable to maintain stable trajectories and do not reach the detector or collision cell [1]. The quadrupole filter is frequently used as mass filter device

Another largely employed type of analyzers is the quadrupole ion trap. The quadrupole ion trap devices are found as two-dimension (2D) also known as linear traps, or three dimen‐ sion (3D) assembly. In case of 2D traps, ions are confined radially by a two-dimensional ra‐ dio frequency field, and axially by stopping potentials applied to end electrodes. It traps the ions in a two dimensional field. When compared to 3D traps [43], linear traps have higher

high vacuum [41].

end electrodes.

*2.2.1. Quadrupole mass filter*

**Figure 1.** A simplified representation of electrospray ionization (ESI) is represented in **A**. The positively charged drop‐ lets and desolvated peptides are represented by the signal +. The superficial tension tends to pull back the charged droplets to the column, while the voltage applied pushes the drop away from the column. The solvent evaporates and the ionized peptides reach the skimmer and go to further analysis. In **B**, the ionization process by matrix-assisted laser desorption/ionization (MALDI) is represented. The laser pulse strikes the MALDI plate, in which the sample is (yellow circles) is co-crystallized with the matrix (grey circles). Either matrix or sample can reach the detector.

### **2.2. Analyzers**

Once the sample is ionized, it enters the mass spectrometer itself. The mass analyzers ex‐ plore different characteristics of the parent or fragmented ions. In case of tandem experi‐ ments, the parent ion must be selected for further fragmentation, and the generated fragment ions will be detected. The combination of two or more analyzers in the same mass spectrometer yielded the high performance and resolution of the nowadays equipments. These characteristics will be briefly detailed ahead.

The main function of a mass analyzer is to separate the ions according to their *m/z* ratio [38], basically by their behavior in electric or magnetic fields [39]. Nowadays, there are few types of analyzers widely used analyzers in tandem mass spectrometry experiments, for proteo‐ mics analyses: quadrupole, quadrupole ion trap, time of flight and orbitrap. These analyzers vary in terms of size, price, resolution, mass range, and the ability to perform tandem mass spectrometry experiments.

In case the ions are separated "in space" (time of flight, TOF; sector; quadrupoles), the tech‐ niques are called beam techniques, because the ions "travel" across the analyzer in a "pulsed beam" mode. In this case, the MS or MS/MS analyses are performed in separated events. The ion trap analyzers, such as quadruploe ion traps and orbitraps characterize ions based on the frequency of their motion in a defined space [15]. Thus MS and MS/MS events can be performed in the same analyzer, being separated by time and not by "space" [15,40]. The an‐ alyzers use magnetic or electric fields, or even combinations of both to select ions. To avoid undesired collisions with neutral gases during analyses, the operation is performed under high vacuum [41].

### *2.2.1. Quadrupole mass filter*

**Figure 1.** A simplified representation of electrospray ionization (ESI) is represented in **A**. The positively charged drop‐ lets and desolvated peptides are represented by the signal +. The superficial tension tends to pull back the charged droplets to the column, while the voltage applied pushes the drop away from the column. The solvent evaporates and the ionized peptides reach the skimmer and go to further analysis. In **B**, the ionization process by matrix-assisted laser desorption/ionization (MALDI) is represented. The laser pulse strikes the MALDI plate, in which the sample is (yellow

Once the sample is ionized, it enters the mass spectrometer itself. The mass analyzers ex‐ plore different characteristics of the parent or fragmented ions. In case of tandem experi‐ ments, the parent ion must be selected for further fragmentation, and the generated fragment ions will be detected. The combination of two or more analyzers in the same mass spectrometer yielded the high performance and resolution of the nowadays equipments.

The main function of a mass analyzer is to separate the ions according to their *m/z* ratio [38], basically by their behavior in electric or magnetic fields [39]. Nowadays, there are few types of analyzers widely used analyzers in tandem mass spectrometry experiments, for proteo‐ mics analyses: quadrupole, quadrupole ion trap, time of flight and orbitrap. These analyzers vary in terms of size, price, resolution, mass range, and the ability to perform tandem mass

circles) is co-crystallized with the matrix (grey circles). Either matrix or sample can reach the detector.

These characteristics will be briefly detailed ahead.

44 Tandem Mass Spectrometry - Molecular Characterization

**2.2. Analyzers**

spectrometry experiments.

A quadrupole analyzer can work as a linear ion trap, in which ions are confined radially by a two-dimensional (2D) radio frequency field, and axially by stopping potentials applied to end electrodes.

A quadrupole mass analyzer is widely used as a "filter" prior fragmentation of the desired ions. Basically it consists of four roods assembled in two pairs, as shown in figure 2A. The first two opposite roods have the same applied voltage, which is different from that of the second two opposite roods establishing a two dimensional quadrupole field in the *x-y* plane (Figure 2A) [38]. The mass analysis depends on the radio frequency, and direct current vol‐ tages which are applied to the four roods [1]. Due to that reason, ions travelling in the quad‐ rupole during analysis will be, at the same time, attracted by one set of roods and repulsed by the second set of roods. Considering the ion population being injected in a quadrupole analyzer, a selection can be made according to their *m/z* ratio, making some ions to have a stable trajectory in the analyzer, while a considerable number of other ions will not go all the way through (Figure 2A, arrows). The ion path occurs in the *z* direction, while the attraction and repulsion are occurring simultaneously in the *x* and *y* direction (Figure 2A) [38,42]. If the oscillations of an ion are stable, the ion will continue to drift down the rod assembly and reach the detector. The stable ions which "travel" all the analyzer length will go to the next steps, which can be detection, fragmentation and a second round of analysis.

The above explanation is quite simple for a complex situation. In a quadrupole mass filter ions of a single *m/z* maintain stable trajectories from the ion source to the detector, whereas ions with different *m/z* values are unable to maintain stable trajectories and do not reach the detector or collision cell [1]. The quadrupole filter is frequently used as mass filter device prior fragmentation in the collision cell, in the case of MS/MS analysis.

### *2.2.2. Quadrupole ion trap analyzers*

Another largely employed type of analyzers is the quadrupole ion trap. The quadrupole ion trap devices are found as two-dimension (2D) also known as linear traps, or three dimen‐ sion (3D) assembly. In case of 2D traps, ions are confined radially by a two-dimensional ra‐ dio frequency field, and axially by stopping potentials applied to end electrodes. It traps the ions in a two dimensional field. When compared to 3D traps [43], linear traps have higher injection efficiencies and higher ion storage capacities [38]. Besides storing ions, they can be combined with other mass analyzers in hybrid instruments and used to isolate ions of select‐ ed mass to charge ratios, to perform tandem mass spectrometry experiments [38,44].

rated by their TOF reach the detector, and a spectrum is presented. Since MALDI produces

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

47

The TOF analyzer can operate in the linear mode or reflectron mode (presented in Figure 2B). In case of the linear mode, the ions fly in the tube and reach the detector. In case of the reflectron mode, the ions fly towards the reflectron which focuses ions with the same *m/z* values, making these ions reach the detector at the same time. Also, there is an adjustment of the kinetic energy since the ions decelerate and accelerate again inside the reflectron. The results are considerable

A couple of years ago, an orbital analyzer, named as the Orbitrap was introduced [40]. The mechanism of analysis is quite different. In an Orbitrap, quoting Michalski and co-workers [52], "the signal is recorded from the image current produced by ion packets which oscillate around and along the spindle-shaped inner electrode of the trap". An extremely simple rep‐ resentation of this principle is shown in figure 2D. This analyzer traps the ions radially around a central spindle electrode. The ion injection is performed perpendicularly to the

In an Orbitrap, the potential distribution of the field is a combination of quadrupole and log‐ arithmic potentials. There are no magnetic or radio frequency fields, so ion stability is ach‐ ieved exclusively due to ions which orbit around an axial electrode and also perform

The necessity of an external ion storage device prior Orbitrap analysis was pointed by Ma‐ karov when the analyzer was developed [53]. In recent years, the common feature of com‐ mercially available mass spectrometers which use an Orbitrap as analyzer is that the trap is preceded by the C-trap, which is an external injection device [54]. The process of capturing ions in the C-trap following by the injection into the Orbitrap is fast and can be easily inter‐ faced and synchronized to any external device such as a linear ion trap mass spectrometer or directly to an ion source. The process of detection is considerably longer, since the sensi‐ tivity is proportional to the square root of number of detected oscillations [40,53,54]. Since the commercial release of the Orbitrap analyzer, there were several changes to the design of the C-trap and the higher-energy collision induced dissociation, which have improved the

The high resolution achieved by the Orbitrap helped the fast adoption of this kind of analyzer, which is considered easy to use, robust and shows excellent performance capabilities [55].

There is no doubt that the fragmentation step of a precursor ion is a key point in proteomics analyses since it enables analyzes at the MS/MS or MSn levels. In tandem mass spectrometry

*2.3.1. Importance of sample preparation for tandem mass spectrometry in proteomics*

mostly single charged species, the *m/z* values correspond to the mass of the ion [50].

more accurate in the reflectron mode than in the linear mode [23,50,51].

*2.2.4. Orbitrap*

longer axis of the trap (*z* axis) [40].

harmonic oscillations along the electrode [40,53].

efficiency and speed of fragmentation [54].

**2.3. Fragmentation**

In all cases, the ion trap is able to store either positively and negatively charged ions, or ions of one specific polarity [38]. In short words, the operation mode of an ion trap is quite similar to that of a quadrupole mass filter; the key difference is that a linear quadrupole is mainly used as a mass filter while the three-dimensional quadrupole used as an ion trap [38,39,44,45]. As the name says, these analyzers are able to trap ions for a specific period of time or to an "amount" of accumulated ions. The quadrupole ion traps analyzers are the best suitable to miniaturization among all kinds of mass analyzers, mainly because they tolerate higher pressures and can work at lower voltages. However, the extreme precision in manufacturing these devices and the low‐ er trapping capacities, can be pointed as disadvantages [44].

When the voltage is applied to the electrodes in the trap, a "trapping potential" is formed, which keeps the ions inside the trap [38,42]. In a ion trap, the trajectories of trapped ions of consecutive specific *m/z* rations are affected and become unstable when the field within the trap is changed. The ions leave the trap according to their *m/z* ratio and reach the detector [45].

In case of 3D traps (also known as 3D Paul traps [43]), three shaped electrodes (two hyper‐ bolic and practically identical) compose the quadrupole ion trap. A simple representation of a 3D ion-trap analyzer is shown in figure 2C. In the case of the regular 3D ion trap shown in figure 2C, the hyperbolic geometry is advantage of the ion traps is that they are used as stor‐ age chambers, mass analyzers or both [46]. Each of the end-cap electrodes has holes in the center for transmission of electrons and ions. The electrons and ions "entrance" is found in one of the endcaps, while the other one endap is the exit "electrode" through which ions will pass to a detector. The ring electrode has an internal hyperbolical surface and it is positioned symmetrically between the two end-cap electrodes [38,44,47]. These traps have mass selec‐ tive detection, storage and ejection capabilities [38].

### *2.2.3. Time of flight analyzer*

Another kind of widely used analyzer is the time-of-flight, TOF. Theoretically the mass range in a TOF analyzer is unlimited [48]. However, in practice, the range is limited by the loss of control over the kinetic energy and spatial distributions of the ions with increasing mass as they are injected into the acceleration region of the mass spectrometer. Consequent‐ ly, the mass accuracy and resolution decrease as the ion mass increases [48,49]. Compared with quadrupole analyzers, the majority of the ions will reach the detector and the lost of ion will not be as expressive as in quadrupole analyzers [39,45].

In TOF analyzers, the desorbed and ionized molecules are accelerated by an electrostatic field and are then ejected through a flight tube under vacuum. In this tube, smaller ions fly faster than larger ions. The detector measures the time of flight for each particular ion. This time to reach the detector depends on the *m/z* of the molecule being analyzed; theoretically, all ions leave the accelerator chamber with the same kinetic energy, and the time to reach the detector will be dependent on the mass of that particular ion (Figure 2B). The ions sepa‐ rated by their TOF reach the detector, and a spectrum is presented. Since MALDI produces mostly single charged species, the *m/z* values correspond to the mass of the ion [50].

The TOF analyzer can operate in the linear mode or reflectron mode (presented in Figure 2B). In case of the linear mode, the ions fly in the tube and reach the detector. In case of the reflectron mode, the ions fly towards the reflectron which focuses ions with the same *m/z* values, making these ions reach the detector at the same time. Also, there is an adjustment of the kinetic energy since the ions decelerate and accelerate again inside the reflectron. The results are considerable more accurate in the reflectron mode than in the linear mode [23,50,51].

### *2.2.4. Orbitrap*

injection efficiencies and higher ion storage capacities [38]. Besides storing ions, they can be combined with other mass analyzers in hybrid instruments and used to isolate ions of select‐

In all cases, the ion trap is able to store either positively and negatively charged ions, or ions of one specific polarity [38]. In short words, the operation mode of an ion trap is quite similar to that of a quadrupole mass filter; the key difference is that a linear quadrupole is mainly used as a mass filter while the three-dimensional quadrupole used as an ion trap [38,39,44,45]. As the name says, these analyzers are able to trap ions for a specific period of time or to an "amount" of accumulated ions. The quadrupole ion traps analyzers are the best suitable to miniaturization among all kinds of mass analyzers, mainly because they tolerate higher pressures and can work at lower voltages. However, the extreme precision in manufacturing these devices and the low‐

When the voltage is applied to the electrodes in the trap, a "trapping potential" is formed, which keeps the ions inside the trap [38,42]. In a ion trap, the trajectories of trapped ions of consecutive specific *m/z* rations are affected and become unstable when the field within the trap is changed.

In case of 3D traps (also known as 3D Paul traps [43]), three shaped electrodes (two hyper‐ bolic and practically identical) compose the quadrupole ion trap. A simple representation of a 3D ion-trap analyzer is shown in figure 2C. In the case of the regular 3D ion trap shown in figure 2C, the hyperbolic geometry is advantage of the ion traps is that they are used as stor‐ age chambers, mass analyzers or both [46]. Each of the end-cap electrodes has holes in the center for transmission of electrons and ions. The electrons and ions "entrance" is found in one of the endcaps, while the other one endap is the exit "electrode" through which ions will pass to a detector. The ring electrode has an internal hyperbolical surface and it is positioned symmetrically between the two end-cap electrodes [38,44,47]. These traps have mass selec‐

Another kind of widely used analyzer is the time-of-flight, TOF. Theoretically the mass range in a TOF analyzer is unlimited [48]. However, in practice, the range is limited by the loss of control over the kinetic energy and spatial distributions of the ions with increasing mass as they are injected into the acceleration region of the mass spectrometer. Consequent‐ ly, the mass accuracy and resolution decrease as the ion mass increases [48,49]. Compared with quadrupole analyzers, the majority of the ions will reach the detector and the lost of

In TOF analyzers, the desorbed and ionized molecules are accelerated by an electrostatic field and are then ejected through a flight tube under vacuum. In this tube, smaller ions fly faster than larger ions. The detector measures the time of flight for each particular ion. This time to reach the detector depends on the *m/z* of the molecule being analyzed; theoretically, all ions leave the accelerator chamber with the same kinetic energy, and the time to reach the detector will be dependent on the mass of that particular ion (Figure 2B). The ions sepa‐

The ions leave the trap according to their *m/z* ratio and reach the detector [45].

ed mass to charge ratios, to perform tandem mass spectrometry experiments [38,44].

er trapping capacities, can be pointed as disadvantages [44].

46 Tandem Mass Spectrometry - Molecular Characterization

tive detection, storage and ejection capabilities [38].

ion will not be as expressive as in quadrupole analyzers [39,45].

*2.2.3. Time of flight analyzer*

A couple of years ago, an orbital analyzer, named as the Orbitrap was introduced [40]. The mechanism of analysis is quite different. In an Orbitrap, quoting Michalski and co-workers [52], "the signal is recorded from the image current produced by ion packets which oscillate around and along the spindle-shaped inner electrode of the trap". An extremely simple rep‐ resentation of this principle is shown in figure 2D. This analyzer traps the ions radially around a central spindle electrode. The ion injection is performed perpendicularly to the longer axis of the trap (*z* axis) [40].

In an Orbitrap, the potential distribution of the field is a combination of quadrupole and log‐ arithmic potentials. There are no magnetic or radio frequency fields, so ion stability is ach‐ ieved exclusively due to ions which orbit around an axial electrode and also perform harmonic oscillations along the electrode [40,53].

The necessity of an external ion storage device prior Orbitrap analysis was pointed by Ma‐ karov when the analyzer was developed [53]. In recent years, the common feature of com‐ mercially available mass spectrometers which use an Orbitrap as analyzer is that the trap is preceded by the C-trap, which is an external injection device [54]. The process of capturing ions in the C-trap following by the injection into the Orbitrap is fast and can be easily inter‐ faced and synchronized to any external device such as a linear ion trap mass spectrometer or directly to an ion source. The process of detection is considerably longer, since the sensi‐ tivity is proportional to the square root of number of detected oscillations [40,53,54]. Since the commercial release of the Orbitrap analyzer, there were several changes to the design of the C-trap and the higher-energy collision induced dissociation, which have improved the efficiency and speed of fragmentation [54].

The high resolution achieved by the Orbitrap helped the fast adoption of this kind of analyzer, which is considered easy to use, robust and shows excellent performance capabilities [55].

### **2.3. Fragmentation**

### *2.3.1. Importance of sample preparation for tandem mass spectrometry in proteomics*

There is no doubt that the fragmentation step of a precursor ion is a key point in proteomics analyses since it enables analyzes at the MS/MS or MSn levels. In tandem mass spectrometry analyses, the first analyzer selects the ion(s) which proceeds to a subsequent section, where the excitation and dissociation steps will happen. Tandem mass spectrometry analyses are the result of two or more sequential separations of ions usually coupling two or more mass analyzers [1,39].

tography tandem mass spectrometry approach, the peptides from the digested proteins are separated by reverse phase chromatography, ionized by ESI and analyzed by tandem mass spectrometry. Abundant proteins will produce abundant peptides, in most cases. These pep‐ tides will be selected for fragmentation regardless the approach, and will mask the low abundant proteins. Besides different strategies used to deal with this situation (exclusion lists, dynamic exclusion, affinity columns), it is still challenging and will not be focused in this chapter. For further reading, please refer to selected references cited ahead [7-10]. The last step is to combine the peptide identification results into a list of proteins that are most

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

49

The most used fragmentation methods used in proteomics analyses are briefly discussed

In collision induced dissociation, also known by collision activated dissociation, the excita‐ tion of the precursor ion is achieved by energetic collisions with an inert gas, usually helium or argon [59]. In CID, the ions selected in the first analyzer are focused in a reaction or colli‐ sion cell, which in several cases is a quadrupole [52,60], prior the reaction in gas phase. In case of peptides, precursor ions are dissociated into fragments along the backbone cleaving at the amide bonds [59,61]. Activation of peptides under low-energy collision conditions

Generally, collisions between the precursor ion and the target gas are accompanied by an increase in the internal energy, which induces decomposition with high probability of frag‐ mentation [62]. The overall process supplies sufficient internal energy to induce covalent bond breakage, and the preferred sites of protonation are the amide bonds of the peptide backbone [62,63]. The protonated amide linkages are weakened and favored to create a ser‐ ies of homologous products ions upon collision-activation. Fragmentation of peptides by CID creates the complementary *b* (in case the charge is retained in the *C*-terminus) and *y* (in

The overall CID process can be divided in two steps: the excitation of the precursor ions and their fragmentation/dissociation. The fragmentation of a precursor ion can occur if the colli‐ sion energy is sufficiently high that the ion is excited beyond its threshold for dissociation. The CID processes can be separated in low and high energy-collisions. Usually, low energy collisions (1-100 eV) is used for organic compounds of moderate masses (hundreds of dal‐ tons), while high energy collision (keV range) are produced in TOF/TOF instruments and the CID spectra resulted from low and high energy collisions are considerably different [62].

There are a number of proteins for which the digestion by trypsin will not be a good choice either because it produces too short or too long peptides. In both cases, CID fragmentation will not be effective for them. Collision-induced dissociation is very effective for short and low charged tryptic peptides, usually less than 20 residues and no more that 4 charges [65,66]. In case of posttranslational modification studies, CID helps mostly to identify the sites and types of the modification (such as phosphorylation, acetylation, etc) in a particular

likely present in the sample [56,58].

*2.3.2. Collision Induced Dissociation - CID*

happens mainly by charge directed reactions [59].

case the charge is retained in the *N*-terminus) ions [64].

ahead.

**Figure 2.** In **A**, there is a representation of a quadrupole mass filter. The ions generated in the ion source migrate through the quadrupole in the *z* direction and at the same time are exposed to simultaneous attraction and repulsion by opposite roods. Ions with stable trajectory are separated in the analyzer and travel all way across (ion 2, green). However, some ions with unstable trajectory are ejected before (ion 1, dark blue). In **B**, the ionization by electrospray and the quadrupole (two roods are shown) and time of flight analyzer is represented. Products from precursor ions previously analyzed and selected for fragmentation (ion 2, green) are accelerated and analyzed in the TOF analyzer. The analyzer might have the reflectron mode or not. The time that the ions spend to travel all way into the TOF is measured and spectrum is recorded. In **C**, the quadrupole ion trap is represented. Ions are trapped for a selected peri‐ od of time or "amount of ions" before being released. A simple representation of the orbitrap analyzer is presented in **D** in which ions are separated based on their orbital movement around the trap.

A typical workflow for protein identification based on tandem mass spectrometry can be divided in three main steps: reduce sample complexity, perform the mass spectrometry analysis and search the data collected against a protein database. First, it is necessary to re‐ duce complexity of a crude biological sample: several strategies can be used in this step [56]. At the protein level it may include organelle fractionation, protein enrichment by immuno‐ percipitation, removal of abundant proteins and fractionation by sodium dodecyl sulfate polyacrylamide electrophoresis. Multidimensional chromatography is widely used at the peptide at level [57]. The digestion of the proteins present in the sample by a peptidase can be performed in solution or in gel after SDS-PAGE fractionation. In case of liquid chroma‐ tography tandem mass spectrometry approach, the peptides from the digested proteins are separated by reverse phase chromatography, ionized by ESI and analyzed by tandem mass spectrometry. Abundant proteins will produce abundant peptides, in most cases. These pep‐ tides will be selected for fragmentation regardless the approach, and will mask the low abundant proteins. Besides different strategies used to deal with this situation (exclusion lists, dynamic exclusion, affinity columns), it is still challenging and will not be focused in this chapter. For further reading, please refer to selected references cited ahead [7-10]. The last step is to combine the peptide identification results into a list of proteins that are most likely present in the sample [56,58].

The most used fragmentation methods used in proteomics analyses are briefly discussed ahead.

### *2.3.2. Collision Induced Dissociation - CID*

analyses, the first analyzer selects the ion(s) which proceeds to a subsequent section, where the excitation and dissociation steps will happen. Tandem mass spectrometry analyses are the result of two or more sequential separations of ions usually coupling two or more mass

**Figure 2.** In **A**, there is a representation of a quadrupole mass filter. The ions generated in the ion source migrate through the quadrupole in the *z* direction and at the same time are exposed to simultaneous attraction and repulsion by opposite roods. Ions with stable trajectory are separated in the analyzer and travel all way across (ion 2, green). However, some ions with unstable trajectory are ejected before (ion 1, dark blue). In **B**, the ionization by electrospray and the quadrupole (two roods are shown) and time of flight analyzer is represented. Products from precursor ions previously analyzed and selected for fragmentation (ion 2, green) are accelerated and analyzed in the TOF analyzer. The analyzer might have the reflectron mode or not. The time that the ions spend to travel all way into the TOF is measured and spectrum is recorded. In **C**, the quadrupole ion trap is represented. Ions are trapped for a selected peri‐ od of time or "amount of ions" before being released. A simple representation of the orbitrap analyzer is presented in

A typical workflow for protein identification based on tandem mass spectrometry can be divided in three main steps: reduce sample complexity, perform the mass spectrometry analysis and search the data collected against a protein database. First, it is necessary to re‐ duce complexity of a crude biological sample: several strategies can be used in this step [56]. At the protein level it may include organelle fractionation, protein enrichment by immuno‐ percipitation, removal of abundant proteins and fractionation by sodium dodecyl sulfate polyacrylamide electrophoresis. Multidimensional chromatography is widely used at the peptide at level [57]. The digestion of the proteins present in the sample by a peptidase can be performed in solution or in gel after SDS-PAGE fractionation. In case of liquid chroma‐

**D** in which ions are separated based on their orbital movement around the trap.

analyzers [1,39].

48 Tandem Mass Spectrometry - Molecular Characterization

In collision induced dissociation, also known by collision activated dissociation, the excita‐ tion of the precursor ion is achieved by energetic collisions with an inert gas, usually helium or argon [59]. In CID, the ions selected in the first analyzer are focused in a reaction or colli‐ sion cell, which in several cases is a quadrupole [52,60], prior the reaction in gas phase. In case of peptides, precursor ions are dissociated into fragments along the backbone cleaving at the amide bonds [59,61]. Activation of peptides under low-energy collision conditions happens mainly by charge directed reactions [59].

Generally, collisions between the precursor ion and the target gas are accompanied by an increase in the internal energy, which induces decomposition with high probability of frag‐ mentation [62]. The overall process supplies sufficient internal energy to induce covalent bond breakage, and the preferred sites of protonation are the amide bonds of the peptide backbone [62,63]. The protonated amide linkages are weakened and favored to create a ser‐ ies of homologous products ions upon collision-activation. Fragmentation of peptides by CID creates the complementary *b* (in case the charge is retained in the *C*-terminus) and *y* (in case the charge is retained in the *N*-terminus) ions [64].

The overall CID process can be divided in two steps: the excitation of the precursor ions and their fragmentation/dissociation. The fragmentation of a precursor ion can occur if the colli‐ sion energy is sufficiently high that the ion is excited beyond its threshold for dissociation. The CID processes can be separated in low and high energy-collisions. Usually, low energy collisions (1-100 eV) is used for organic compounds of moderate masses (hundreds of dal‐ tons), while high energy collision (keV range) are produced in TOF/TOF instruments and the CID spectra resulted from low and high energy collisions are considerably different [62].

There are a number of proteins for which the digestion by trypsin will not be a good choice either because it produces too short or too long peptides. In both cases, CID fragmentation will not be effective for them. Collision-induced dissociation is very effective for short and low charged tryptic peptides, usually less than 20 residues and no more that 4 charges [65,66]. In case of posttranslational modification studies, CID helps mostly to identify the sites and types of the modification (such as phosphorylation, acetylation, etc) in a particular protein [66,67]. Even though the sequence of the protein is known, in complexes samples they are not pure. For this reason, individual digestion of the same by two different pepti‐ dases and analyses by different fragmentation methods in tandem mass spectrometry ap‐ proaches is used.

backbone and will produce the *c*- and *z*-product ions [64,66,68]. As ECD, ETD prevents frag‐

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

51

The ion/ion chemistry in EDT requires few milliseconds to be completed. Besides that, ETD can be performed with femtomole amount of sample and on a compatible timescale to liq‐ uid chromatography and MS analyses. Also, ETD is advantageous in the study of larger peptides, which carry three or more charges. These larger and highly charged peptides offer favorable cleavage conditions during ETD [66]. However, it is important to remember that when working with ETD, a different protease must be used, instead of trypsin, to produce larger peptides (25 aminoacids or more). Longer peptides tend to gain more charges (13 to 16) based on the increased number of basic residues [66]. Endopeptidases Lys-C and Asp-N usually yield longer peptides, and make a good choice with ETD fragmentation [66,73].

The main idea of this chapter was to provide an overview on the most used components/ methods in proteomics analyses employing mass spectrometer. A brief overview on the ion‐ ization methods, analyzers and fragmentation was given and can be used as a support for

The author´s research is supported in part by CNPq, CAPES and FAPERGS (Brazil). The author would like to express his gratitude to Dr. Celia Carlini, for critical reading of this chapter.

Department of Biophysics and Center of Biotechnology – Universidade Federal do Rio

[1] Glish GL, Vachet RW. The Basics of Mass Spectrometry in the Twenty-First Century.

ile posttranslational modifications from fragmentation [64,66].

**3. Concluding remarks**

new researches on the field.

**Acknowledgements**

**Author details**

**References**

Diogo Ribeiro Demartini

Address all correspondence to: diogord@terra.com.br

Nature Reviews Drug Discovery 2003;2(2) 140-150.

Grande do Sul, Porto Alegre, RS, Brazil

### *2.3.3. Electron Capture Dissociation - ECD*

Electron capture dissociation is based on the dissociative recombination of multiply proto‐ nated polypeptide molecules with low-energy electrons [68]. The mechanism of ECD is not completely understood, however, in general terms, can be explained as follow. Polypeptide polycations initially capture an electron in a high orbit, followed by a charge neutralization, which leads to an excited radical species that undergoes bond cleavage [64,68]. The radical species dissociates through N–Cα bond cleavages to produce *c-* and *z*-type product ions. The mechanism (s) by which the N–Cα bond cleavages occur is believed that following elec‐ tron capture, a hydrogen atom is transferred to an amide carbonyl [68-70] or a proton is transferred to an amide anion radical [68,71], both resulting in the formation of an aminoke‐ tyl radical intermediate, which dissociates through N–Cα bond cleavages.

Fragmentation in ECD happens at a very high rate, however, the time required for electron cap‐ ture by precursor ions exceeds the residence time in most mass spectrometers, such as those with TOF and quadrupole analyzers. Another important point is that ECD efficiency is highest for low energy electrons [69], which is difficult to provide in ion traps, for example. For that rea‐ son, ECD is mostly used in Fourier transform ion cyclotron resonance mass spectrometry [68].

Electron capture dissociation has been shown advantageous in sequence characterization, *de novo* sequencing, disulphide bond analysis [69,71], and posttranslational modification analy‐ ses [68]. The information obtained by ECD is complementary to those obtained by CID in traditional MS/MS methods [68-70]. One important advantage of ECD is to promote a lower energy pathway than CID; even though fragmentation happens, fragile posttranslational modifications are preserved [66].

### *2.3.4. Electron Transfer Dissociation - ETD*

The electron-transfer dissociation (ETD) is based on ion/ion chemistry [66,72,73]. It involves transferring an electron from a radical anion to a protonated peptide [72,73], resulting in cleavages at the N-Cα bond of the peptide backbone and preserving most of the posttransla‐ tional modifications [66]. Differently from CID, but similar to ECD, ETD creates the comple‐ mentary *c-* and *z*-type ions. The ETD is widely used for posttranslational modification studies, such as phosphorylation specially because the predominant loss of the phosphate moiety by neutral loss generally precludes further fragmentation.

Different from ECD, in which the primary source of excess energy is the recombination en‐ ergy released when the electron is captured, in EDT this recombination energy is reduced by the electron binding energy to the anion donor; ETD involves transfer of an electron to the multiply protonated precursor ion from a singly charged radical [64,71]. However, similar to ECD, ETD also induces relatively non-selective cleavage of the N–Cα bond on a peptide's backbone and will produce the *c*- and *z*-product ions [64,66,68]. As ECD, ETD prevents frag‐ ile posttranslational modifications from fragmentation [64,66].

The ion/ion chemistry in EDT requires few milliseconds to be completed. Besides that, ETD can be performed with femtomole amount of sample and on a compatible timescale to liq‐ uid chromatography and MS analyses. Also, ETD is advantageous in the study of larger peptides, which carry three or more charges. These larger and highly charged peptides offer favorable cleavage conditions during ETD [66]. However, it is important to remember that when working with ETD, a different protease must be used, instead of trypsin, to produce larger peptides (25 aminoacids or more). Longer peptides tend to gain more charges (13 to 16) based on the increased number of basic residues [66]. Endopeptidases Lys-C and Asp-N usually yield longer peptides, and make a good choice with ETD fragmentation [66,73].

### **3. Concluding remarks**

protein [66,67]. Even though the sequence of the protein is known, in complexes samples they are not pure. For this reason, individual digestion of the same by two different pepti‐ dases and analyses by different fragmentation methods in tandem mass spectrometry ap‐

Electron capture dissociation is based on the dissociative recombination of multiply proto‐ nated polypeptide molecules with low-energy electrons [68]. The mechanism of ECD is not completely understood, however, in general terms, can be explained as follow. Polypeptide polycations initially capture an electron in a high orbit, followed by a charge neutralization, which leads to an excited radical species that undergoes bond cleavage [64,68]. The radical species dissociates through N–Cα bond cleavages to produce *c-* and *z*-type product ions. The mechanism (s) by which the N–Cα bond cleavages occur is believed that following elec‐ tron capture, a hydrogen atom is transferred to an amide carbonyl [68-70] or a proton is transferred to an amide anion radical [68,71], both resulting in the formation of an aminoke‐

Fragmentation in ECD happens at a very high rate, however, the time required for electron cap‐ ture by precursor ions exceeds the residence time in most mass spectrometers, such as those with TOF and quadrupole analyzers. Another important point is that ECD efficiency is highest for low energy electrons [69], which is difficult to provide in ion traps, for example. For that rea‐ son, ECD is mostly used in Fourier transform ion cyclotron resonance mass spectrometry [68].

Electron capture dissociation has been shown advantageous in sequence characterization, *de novo* sequencing, disulphide bond analysis [69,71], and posttranslational modification analy‐ ses [68]. The information obtained by ECD is complementary to those obtained by CID in traditional MS/MS methods [68-70]. One important advantage of ECD is to promote a lower energy pathway than CID; even though fragmentation happens, fragile posttranslational

The electron-transfer dissociation (ETD) is based on ion/ion chemistry [66,72,73]. It involves transferring an electron from a radical anion to a protonated peptide [72,73], resulting in cleavages at the N-Cα bond of the peptide backbone and preserving most of the posttransla‐ tional modifications [66]. Differently from CID, but similar to ECD, ETD creates the comple‐ mentary *c-* and *z*-type ions. The ETD is widely used for posttranslational modification studies, such as phosphorylation specially because the predominant loss of the phosphate

Different from ECD, in which the primary source of excess energy is the recombination en‐ ergy released when the electron is captured, in EDT this recombination energy is reduced by the electron binding energy to the anion donor; ETD involves transfer of an electron to the multiply protonated precursor ion from a singly charged radical [64,71]. However, similar to ECD, ETD also induces relatively non-selective cleavage of the N–Cα bond on a peptide's

moiety by neutral loss generally precludes further fragmentation.

tyl radical intermediate, which dissociates through N–Cα bond cleavages.

proaches is used.

*2.3.3. Electron Capture Dissociation - ECD*

50 Tandem Mass Spectrometry - Molecular Characterization

modifications are preserved [66].

*2.3.4. Electron Transfer Dissociation - ETD*

The main idea of this chapter was to provide an overview on the most used components/ methods in proteomics analyses employing mass spectrometer. A brief overview on the ion‐ ization methods, analyzers and fragmentation was given and can be used as a support for new researches on the field.

### **Acknowledgements**

The author´s research is supported in part by CNPq, CAPES and FAPERGS (Brazil). The author would like to express his gratitude to Dr. Celia Carlini, for critical reading of this chapter.

### **Author details**

Diogo Ribeiro Demartini

Address all correspondence to: diogord@terra.com.br

Department of Biophysics and Center of Biotechnology – Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

### **References**

[1] Glish GL, Vachet RW. The Basics of Mass Spectrometry in the Twenty-First Century. Nature Reviews Drug Discovery 2003;2(2) 140-150.

[2] Ong SE, Mann M. Mass Spectrometry-Based Proteomics Turns Quantitative. Nature Chemical Biology 2005;1(5) 252-262.

[14] Wilm M. Principles of Electrospray Ionization. Molecular & Cellular Proteomics:

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

53

[15] Glish GL, Burinsky DJ. Hybrid Mass Spectrometers for Tandem Mass Spectrometry. Journal of the American Society for Mass Spectrometry 2008;19(2) 161-172.

[16] Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray Ionization for

[17] Gabelica V, De PE. Internal Energy and Fragmentation of Ions Produced in Electro‐

[18] Goodlett DR, Yi EC. Proteomics Without Polyacrylamide: Qualitative and Quantita‐ tive Uses of Tandem Mass Spectrometry in Proteome Analysis. Functional and Inte‐

[19] Chen P. Electrospray Ionization Tandem Mass Spectrometry in High-Throughput Screening of Homogeneous Catalysts. Angewandte Chemie - International Edition

[20] Murphy RC, Barkley RM, Berry KZ, Hankin J, Harrison K, Johnson C, Krank J, McA‐ noy A, Uhlson C, Zarini S. Electrospray Ionization and Tandem Mass Spectrometry

[21] Iribarne JV, Dziedzic PJ, Thomson BA. Atmospheric Pressure Ion Evaporation-Mass Spectrometry. International Journal of Mass Spectrometry and Ion Physics 1983;50(3)

[22] Schmelzeisen-Redeker G, Bütfering L, Röllgen FW. Desolvation of Ions and Mole‐ cules in Thermospray Mass Spectrometry. International Journal of Mass Spectrome‐

[23] Wieser A, Schneider L, Jung J, Schubert S. MALDI-TOF MS in Microbiological Diag‐ nostics-Identification of Microorganisms and Beyond (Mini Review). Applied Micro‐

[24] Dreisewerd K. The Desorption Process in MALDI. Chemical Reviews 2003;103(2)

[25] Zenobi R, Knochenmuss R. Ion Formation in MALDI Mass Spectrometry. Mass Spec‐

[26] Knochenmuss R, Zenobi R. MALDI Ionization: the Role of in-Plume Processes.

[27] Zhigilei LV, Garrison BJ. Velocity Distributions of Analyte Molecules in Matrix-As‐ sisted Laser Desorption From Computer Simulations. Rapid Communications in

[28] Zhigilei LV, Garrison BJ. Velocity Distributions of Molecules Ejected in Laser Abla‐

Mass Spectrometry of Large Biomolecules. Science 1989;246(4926) 64-71.

spray Sources. Mass Spectrometry Reviews 2005;24(4) 566-587.

of Eicosanoids. Analytical Biochemistry 2005;346(1) 1-42.

grative Genomics 2002;2(4-5) 138-153.

try and Ion Processes 1989;90(2) 139-150.

trometry Reviews 1998;17(5) 337-366.

Chemical Reviews 2003;103(2) 441-452.

Mass Spectrometry 1998;12(18) 1273-1277.

tion. Applied Physics Letters 1997;71(4) 551-553.

biology and Biotechnology 2012;93(3) 965-974.

2003;42(25) 2832-2847.

331-347.

395-426.

MCP 2011;10(7) 1-8.


[14] Wilm M. Principles of Electrospray Ionization. Molecular & Cellular Proteomics: MCP 2011;10(7) 1-8.

[2] Ong SE, Mann M. Mass Spectrometry-Based Proteomics Turns Quantitative. Nature

[3] Wong JW, Cagney G. An Overview of Label-Free Quantitation Methods in Proteo‐ mics by Mass Spectrometry. Methods in Molecular Biology (Clifton, N. J. )

[4] Aebersold R, Mann M. Mass Spectrometry-Based Proteomics. Nature 2003;422(6928)

[5] Ishihama Y. Proteomic LC-MS Systems Using Nanoscale Liquid Chromatography With Tandem Mass Spectrometry. Journal of Chromatography. A 2005;1067(1-2)

[6] Granvogl B, Plöcher M, Eichacker LA. Sample Preparation by in-Gel Digestion for Mass Spectrometry-Based Proteomics. Analytical and Bioanalytical Chemistry

[7] Fong KP, Barry C, Tran AN, Traxler EA, Wannemacher KM, Tang HY, Speicher KD, Blair IA, Speicher DW, Grosser T, Brass LF. Deciphering the Human Platelet Shed‐

[8] Muntel J, Hecker M, Becher D. An Exclusion List Based Label-Free Proteome Quanti‐ fication Approach Using an LTQ Orbitrap. Rapid Communications in Mass Spec‐

[9] Zhang Y, Wen Z, Washburn MP, Florens L. Effect of Dynamic Exclusion Duration on Spectral Count Based Quantitative Proteomics. Analytical Chemistry 2009;81(15)

[10] Ferro M, Brugiere S, Salvi D, Seigneurin-Berny D, Court M, Moyet L, Ramus C, Miras S, Mellal M, Le GS, Kieffer-Jaquinod S, Bruley C, Garin J, Joyard J, Masselon C, Roll‐ and N. AT\_CHLORO: A Comprehensive Chloroplast Proteome Database With Sub‐ plastidiallocalization and Curated Information on Envelope Proteins. Molecular &

[11] Seigneurin-Berny D, Rolland N, Garin J, Joyard J. Differential Extraction of Hydro‐ phobic Proteins From Chloroplast Envelope Membranes: A Subcellular-Specific Pro‐ teomic Approach to Identify Rare Intrinsic Membrane Proteins. Plant Journal

[12] Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B. Quantitative Mass Spectrom‐ etry in Proteomics: a Critical Review. Analytical and Bioanalytical Chemistry

[13] Banerjee S, Mazumdar S. Electrospray Ionization Mass Spectrometry: a Technique to Access the Information Beyond the Molecular Weight of the Analyte. International

Chemical Biology 2005;1(5) 252-262.

52 Tandem Mass Spectrometry - Molecular Characterization

2010;604273-283.

2007;389(4) 991-1002.

dome. Blood 2011;117(1) e15-e26.

Cellular Proteomics: MCP 2010;9(6) 1063-1084.

Journal of Analytical Chemistry 2012;20121-40.

trometry 2012;26(6) 701-709.

6317-6326.

1999;19(2) 217-228.

2007;389(4) 1017-1031.

198-207.

73-83.


[29] Benk AS, Roesli C. Label-Free Quantification Using MALDI Mass Spectrometry: Considerations and Perspectives. Analytical and Bioanalytical Chemistry 2012;404(4) 1039-1056.

[43] Paul W. Electromagnetic Traps for Charged and Neutral Particles. Reviews of Mod‐

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

55

[44] Ouyang Z, Gao L, Fico M, Chappell WJ, Noll RJ, Cooks RG. Quadrupole Ion Traps and Trap Arrays: Geometry, Material, Scale, Performance. European Journal of Mass

[45] March RE. Quadrupole Ion Trap Mass Spectrometry: a View at the Turn of the Cen‐ tury. International Journal of Mass Spectrometry 2000;200(1ΓÇô3) 285-312.

[46] McAlister GC, Phanstiel D, Good DM, Berggren WT, Coon JJ. Implementation of Electron-Transfer Dissociation on a Hybrid Linear Ion Trap-Orbitrap Mass Spectrom‐

[47] Douglas DJ, Frank AJ, Mao D. Linear Ion Traps in Mass Spectrometry. Mass Spec‐

[48] Lee J, Chen H, Liu T, Berkman CE, Reilly PT. High Resolution Time-of-Flight Mass Analysis of the Entire Range of Intact Singly-Charged Proteins. Analytical Chemistry

[49] Lee J, Reilly PTA. Limitation of Time-of-Flight Resolution in the Ultra High Mass

[50] Croxatto A, Prod'hom G, Greub G. Applications of MALDI-TOF Mass Spectrometry in Clinical Diagnostic Microbiology. FEMS Microbiology Reviews 2012;36(2) 380-407.

[51] Bonk T, Humeny A. MALDI-TOF-MS Analysis of Protein and DNA. Neuroscientist.

[52] Michalski A, Damoc E, Lange O, Denisov E, Nolting D, Muller M, Viner R, Schwartz J, Remes P, Belford M, Dunyach JJ, Cox J, Horning S, Mann M, Makarov A. Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facili‐ tates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes. Molecular

[53] Makarov A. Electrostatic Axially Harmonic Orbital Trapping: a High-Performance

[54] Makarov A, Scigelova M. Coupling Liquid Chromatography to Orbitrap Mass Spec‐

[55] Kelstrup CD, Young C, Lavallee R, Nielsen ML, Olsen JV. Optimized Fast and Sensi‐ tive Acquisition Methods for Shotgun Proteomics on a Quadrupole Orbitrap Mass

[56] Hernandez P, Muller M, Appel RD. Automated Protein Identification by Tandem Mass Spectrometry: Issues and Strategies. Mass Spectrometry Reviews 2006;25(2)

Technique of Mass Analysis. Analytical Chemistry 2000;72(6) 1156-1162.

trometry. Journal of Chromatography. A 2010;1217(25) 3938-3945.

Spectrometer. Journal of Proteome Research 2012;11(6) 3487-3497.

ern Physics 1990;62(3) 531-540.

trometry Reviews 2005;24(1) 1-29.

2011;83(24) 9406-9412.

2001;7(1) .

235-254.

Spectrometry (Chichester, England) 2007;13(1) 13-18.

eter. Analytical Chemistry 2007;79(10) 3525-3534.

Range. Analytical Chemistry 2011;83(15) 5831-5833.

& Cellular Proteomics: MCP 2012;11(3) O111.


[43] Paul W. Electromagnetic Traps for Charged and Neutral Particles. Reviews of Mod‐ ern Physics 1990;62(3) 531-540.

[29] Benk AS, Roesli C. Label-Free Quantification Using MALDI Mass Spectrometry: Considerations and Perspectives. Analytical and Bioanalytical Chemistry 2012;404(4)

[30] Reyzer ML, Caprioli RM. MALDI Mass Spectrometry for Direct Tissue Analysis: a New Tool for Biomarker Discovery. Journal of Proteome Research 2005;4(4)

[31] Trimpin S, Inutan ED, Herath TN, McEwen CN. Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Method for Selectively Producing Either Singly or

[32] Suckau D, Resemann A, Schuerenberg M, Hufnagel P, Franzen J, Holle A. A Novel MALDI LIFT-TOF/TOF Mass Spectrometer for Proteomics. Analytical and Bioanalyt‐

[33] Smith SA, Blake TA, Ifa DR, Cooks RG, Ouyang Z. Dual-Source Mass Spectrometer With MALDI-LIT-ESI Configuration. Journal of Proteome Research 2007;6(2) 837-845.

[34] Debois D, Smargiasso N, Demeure K, Asakawa D, Zimmerman TA, Quinton L, De PE. MALDI In-Source Decay, From Sequencing to Imaging. Topics in Current Chem‐

[35] Hillenkamp F, Karas M. The MALDI Process and Method. In: MALDI MS. Wiley-

[36] Tholey A, Heinzle E. Ionic (Liquid) Matrices for Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry-Applications and Perspectives. Analytical and Bioana‐

[37] Chang WC, Huang LC, Wang YS, Peng WP, Chang HC, Hsu NY, Yang WB, Chen CH. Matrix-Assisted Laser Desorption/Ionization (MALDI) Mechanism Revisited.

[38] March RE. Quadrupole Ion Traps. Mass Spectrometry Reviews 2009;28(6) 961-989.

[39] El-Aneed A, Cohen A, Banoub J. Mass Spectrometry, Review of the Basics: Electro‐ spray, MALDI, and Commonly Used Mass Analyzers. Applied Spectroscopy Re‐

[40] Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Graham CR. The Orbitrap: a New

[41] Schaeffer-Reiss C. A Brief Summary of the Different Types of Mass Spectrometers Used in Proteomics. Methods in Molecular Biology (Clifton, N. J. ) 2008;4843-16.

[42] Douglas DJ, Konenkov NV. Trajectory Calculations of Space-Charge-Induced Mass Shifts in a Linear Quadrupole Ion Trap. Rapid Communications in Mass Spectrome‐

Mass Spectrometer. Journal of Mass Spectrometry 2005;40(4) 430-443.

Multiply Charged Molecular Ions. Analytical Chemistry 2010;82(1) 11-15.

1039-1056.

54 Tandem Mass Spectrometry - Molecular Characterization

1138-1142.

istry 2012.

ical Chemistry 2003;376(7) 952-965.

VCH Verlag GmbH & Co. KGaA; (2007). 1-28

lytical Chemistry 2006;386(1) 24-37.

Analytica Chimica Acta 2007;582(1) 1-9.

views 2009;44(3) 210-230.

try 2012;26(18) 2105-2114.


[57] Washburn MP, Wolters D, Yates JR. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nature Biotechnology 2001;19(3) 242-247.

[71] Simons J. Mechanisms for S-S and N-Cα Bond Cleavage in Peptide ECD and ETD

A Short Overview of the Components in Mass Spectrometry Instrumentation for Proteomics Analyses

http://dx.doi.org/10.5772/54484

57

[72] Kim MS, Pandey A. Electron Transfer Dissociation Mass Spectrometry in Proteomics.

[73] Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF. Peptide and Protein Se‐ quence Analysis by Electron Transfer Dissociation Mass Spectrometry. Proceedings of the National Academy of Sciences of the United States of America 2004;101(26)

Mass Spectrometry. Chemical Physics Letters 2010;484(20) 81-95.

Proteomics 2012;12(4-5) 530-542.

9528-9533.


[71] Simons J. Mechanisms for S-S and N-Cα Bond Cleavage in Peptide ECD and ETD Mass Spectrometry. Chemical Physics Letters 2010;484(20) 81-95.

[57] Washburn MP, Wolters D, Yates JR. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nature Biotechnology

[58] Coon JJ, Syka JEP, Shabanowitz J, Hunt DF. Tandem Mass Spectrometry for Peptide

[59] Paizs B, Suhai S. Fragmentation Pathways of Protonated Peptides. Mass Spectrome‐

[60] Boersema PJ, Mohammed S, Heck AJR. Phosphopeptide Fragmentation and Analysis

[61] Hunt DF, Yates JR, Shabanowitz J, Winston S, Hauer CR. Protein Sequencing by Tan‐ dem Mass Spectrometry. Proceedings of the National Academy of Sciences of the

[62] Sleno L, Volmer DA. Ion Activation Methods for Tandem Mass Spectrometry. Jour‐

[63] Shukla AK, Futrell JH. Tandem Mass Spectrometry: Dissociation of Ions by Collision‐

[64] Palumbo AM, Smith SA, Kalcic CL, Dantus M, Stemmer PM, Reid GE. Tandem Mass Spectrometry Strategies for Phosphoproteome Analysis. Mass Spectrometry Reviews

[65] Mikesh LM, Ueberheide B, Chi A, Coon JJ, Syka JE, Shabanowitz J, Hunt DF. The Utility of ETD Mass Spectrometry in Proteomic Analysis. Biochimica Et Biophysica

[66] Wiesner J, Premsler T, Sickmann A. Application of Electron Transfer Dissociation (ETD) for the Analysis of Posttranslational Modifications. Proteomics 2008;8(21)

[67] Miller K, Hao Z, Zhang T, Huhmer A. Alternating Real-Time Data Dependent ETD and CID for Improved Protein Coverage by LC/MS on a Linear Ion Trap Mass Spec‐

[68] Zubarev RA. Electron-Capture Dissociation Tandem Mass Spectrometry. Current

[69] Kalli A, Hess S. Electron Capture Dissociation of Hydrogen-Deficient Peptide Radi‐ cal Cations. Journal of the American Society for Mass Spectrometry 2012;23(10)

[70] Zubarev RA, Haselmann KF, Budnik B, Kjeldsen F, Jensen F. Towards an Under‐ standing of the Mechanism of Electron-Capture Dissociation: a Historical Perspective

and Modern Ideas. European Journal of Mass Spectrometry 2002;8(5) 337-349.

trometer. Molecular & Cellular Proteomics: MCP 2006;5(10) .

Opinion in Biotechnology 2004;15(1) 12-16.

al Activation. Journal of Mass Spectrometry 2000;35(9) 1069-1090.

by Mass Spectrometry. Journal of Mass Spectrometry 2009;44(6) 861-878.

and Protein Sequence Analysis. Biotechniques 2005;38(4) 519-523.

2001;19(3) 242-247.

56 Tandem Mass Spectrometry - Molecular Characterization

2011;30(4) 600-625.

4466-4483.

1729-1740.

Acta 2006;1764(12) 1811-1822.

try Reviews 2005;24(4) 508-548.

United States of America 1986;83(17) 6233-6237.

nal of Mass Spectrometry 2004;39(10) 1091-1112.


**Section 2**

**Molecular Characterization by Mass**

**Spectrometry**

**Molecular Characterization by Mass Spectrometry**

**Chapter 3**

**Contribution of Mass Spectrometry to the Study of**

Mass spectrometry (MS) has become a powerful analytical tool for qualitative and quantita‐ tive applications, providing information about the structure and purity of compounds, and

The most recent applications of mass spectrometry are oriented towards biochemical appli‐ cations such as proteome, metabolome and drug discovery. During the last decade, mass spectrometry has progressed rapidly and an evolution has been observed in the type of ap‐ plications, software and equipments. Atmospheric pressure ionization sources are now used, an analyser based on a new concept (the orbitrap) was recently developed, existing ones were modified, and new hybrid instruments were developed using combinations of different analysers, depending on applications. One of the major trends was the transition to high resolution/accurate mass analysis, made routine by new MS instruments. The use of separation techniques as gas chromatography (GC), liquid chromatography (LC) and capil‐ lary electrophoresis (CE) coupled with mass spectrometry and tandem mass spectrometry,

In this chapter are presented general aspects related with characteristics of mass spectrome‐ try equipments. The contribution of this technique to new discoveries concerning one of the

Mass spectrometry is a technique used to analyse from small inorganic molecules to biologi‐ cal macromolecules and relies on the formation of gas-phase ions (positively or negatively

> © 2013 Sitoe et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Sitoe et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Antimalarial Agents**

http://dx.doi.org/10.5772/56225

**1. Introduction**

Ana Raquel Sitoe, Francisca Lopes, Rui Moreira,

Additional information is available at the end of the chapter

also about the chemical composition of complex samples.

major infectious diseases in man, malaria, is also discussed.

expanded the interest in this methodology.

**2. Basics of mass spectrometry**

Ana Coelho and Maria Rosário Bronze
