**3.3 Metrology for protein quantification based on mass spectrometry**

Over the last decade, mass spectrometry-based approaches has been shown to be the most powerful tool for protein characterization and become available in many laboratories (Cravatt et al., 2007). Although early biological studies using mass spectrometry merely provided lists of proteins identified in a particular sample, mass spectrometry-based proteomic strategies have been developed, improved, and used for many applications. With the respect to quantitative protein analysis, mass spectrometry alone does not provide much information. MS in general can't detect a whole protein in a quantitative manner. To improve quantification performance, a whole protein needs to be reduced to amino acids or peptides, which are done by chemical or enzymatic hydrolysis, respectively. Even so, repeatability of MS quantification is poor due to the complex nature of ionization prior to MS analysis, which can be dramatically mitigated by the format of isotope dilution-mass spectrometry (IDMS). For the reductive approaches, conversion of the target protein to amino acids or peptides while maintaining the original stoichiometry is a key issue. There could be much debate on the completeness of reduction. This issue is addressed in the following sections. In addition, the sound measurement traceability in quantification of proteins should be established as a very basic requirement for establishment of metrology, which is highly likely to be achieved through preparation for high quality amino acids CRMs for amino acid based quantification. Nevertheless, the establishment of metrology for protein quantification seems to be a doable task based on advanced mass spectrometry, and the refinements of analytical strategies for reduced measurement uncertainties are pursued as below.

#### **3.3.1 Amino acid analysis**

432 Modern Metrology Concerns

There are a number of quantification methods for proteins either bioassays or instrumental methods. The most commonly used methodologies in bioassays are the Biuret (Savory et al., 1968), Bradford (Bradford, 1976), Lowry (Fryer et al., 1986), and bicinchoninic acid (BCA) (Smith et al., 1985) assays. These methods employ chemical reagents which specifically react with proteins to produce colored products which can be measured by UV spectrophotometer in a concentration-dependent manner. The absorbance of colored sample is compared to standard curves constructed with a known protein (frequently bovine serum albumin) in order to determine concentration in unknown samples. Biological methods are widely used owing to their simplicity and low cost, but are hampered by poor accuracy and reproducibility since their responses are either assay or calibrator-protein dependent, and also the results can vary with the residue composition of the target protein. Thus bioassays lead to relative differences in protein quantification and cannot give absolute values. On the other hand, instrumental analytical methods including chromatographic techniques (HPLC, GC), capillary electrophoresis (CE), and mass spectrometry (MS) are often favored because of higher precision. These methods may require time-consuming sample preparation, which leads to higher costs than biological methods. Nevertheless, both bioassay and instrumental analysis need to be calibrated with a highly reliable protein standard material to ensure accurate and comparable results. A higher order analytical method needs to be established

The same is true for high throughput analysis formats such as 2D-PAGE gels (Smithies and Poulik, 1956) and enzyme-lingked immunosorbent assay (ELISA). These methods are particularly well fit the purpose of high throughput analysis for proteomics research and protein-chip analysis, respectively. Therefore, they have become powerful tools for screening effective protein markers. As such research progresses, it has become obvious that reliable quantification in an absolute manner is essentially required for comparability of

Over the last decade, mass spectrometry-based approaches has been shown to be the most powerful tool for protein characterization and become available in many laboratories (Cravatt et al., 2007). Although early biological studies using mass spectrometry merely provided lists of proteins identified in a particular sample, mass spectrometry-based proteomic strategies have been developed, improved, and used for many applications. With the respect to quantitative protein analysis, mass spectrometry alone does not provide much information. MS in general can't detect a whole protein in a quantitative manner. To improve quantification performance, a whole protein needs to be reduced to amino acids or peptides, which are done by chemical or enzymatic hydrolysis, respectively. Even so, repeatability of MS quantification is poor due to the complex nature of ionization prior to MS analysis, which can be dramatically mitigated by the format of isotope dilution-mass spectrometry (IDMS). For the reductive approaches, conversion of the target protein to amino acids or peptides while maintaining the original stoichiometry is a key issue. There could be much debate on the completeness of reduction. This issue is addressed in the following sections. In addition, the sound measurement traceability in quantification of proteins should be established as a very basic requirement for establishment of metrology,

for accurate determination of such protein standard materials.

**3.3 Metrology for protein quantification based on mass spectrometry** 

**3.2 Conventional methods** 

data.

Amino acid analysis has been used in many applications as a conventional protein analysis technique. Until the emergence of mass spectrometry, amino acid analysis was essential to the identification of proteins. Nowadays, the analysis is known to be a powerful tool for determination of protein quantities based on detailed information regarding precise quantification of free amino acids. This quantitative analysis is based on the total amount of single amino acids, so the preparation of a highly pure sample is a prerequisite for an accurate analysis. The analysis consists of three steps; hydrolysis of the sample to amino acid constituents (Fig. 3.1), chromatographic separation of the target amino acid to be analyzed, and quantification by mass spectrometry with a labeled internal standard (Fig. 3.2). Of among, complete hydrolysis is critical for overall accuracy of the procedure (Albin et al., 2000; Anderson et al., 1977; Darragh et al., 1996; Fountoulakis and Lahm, 1998; Kinumi et al., 2010). Moreover, for the analysis of an unknown protein, optimization of hydrolysis conditions should be considered before conducting the whole procedure. Very recently, KRISS scientists have reported quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid chromatography-tandem mass spectrometry (Jeong et al., 2011), which is briefly summarized below.

Fig. 3.1. Acidic hydrolysis for amino acid analysis

1. Sample purity assessment: As contaminant components in a protein sample can lead to overestimation of the target quantities, it is important to determine the precise sample purity. To this end, properly diluted human growth hormone (hGH) solution was subjected to capillary zone electrophoresis (CE). The analysis indicates that no significant impurities were present in the sample. The results were also confirmed by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Fig. 3.2. Schematic of MS based amino acid analysis

2. Hydrolysis: As mentioned previously, optimizing the reaction conditions for protein hydrolysis is critical to minimize analytical variations. Through extensive optimization procedures, we found suitable reaction conditions for acidic hydrolysis with hydrochloric acid (HCl), which is the most common hydrolysis method to date. The experiments were performed to test the effects of various factors, including hydrolysis time, protein concentration, HCl concentration, and hydrolysis temperature. As the internal standards should be spiked to the sample prior to the hydrolysis step, the stability of the isotopically labeled free amino acids was also monitored under the identical reaction conditions. The results indicate that the defined hydrolysis step causes no substantial degradation of the internal standard candidates. Therefore, the hGH sample and isotope labeled amino acids were mixed, hydrolyzed, and subjected to HPLC-MS/MS.

1. Sample purity assessment: As contaminant components in a protein sample can lead to overestimation of the target quantities, it is important to determine the precise sample purity. To this end, properly diluted human growth hormone (hGH) solution was subjected to capillary zone electrophoresis (CE). The analysis indicates that no significant impurities were present in the sample. The results were also confirmed by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption

2. Hydrolysis: As mentioned previously, optimizing the reaction conditions for protein hydrolysis is critical to minimize analytical variations. Through extensive optimization procedures, we found suitable reaction conditions for acidic hydrolysis with hydrochloric acid (HCl), which is the most common hydrolysis method to date. The experiments were performed to test the effects of various factors, including hydrolysis time, protein concentration, HCl concentration, and hydrolysis temperature. As the internal standards should be spiked to the sample prior to the hydrolysis step, the stability of the isotopically labeled free amino acids was also monitored under the identical reaction conditions. The results indicate that the defined hydrolysis step causes no substantial degradation of the internal standard candidates. Therefore, the hGH sample and isotope labeled amino acids were mixed, hydrolyzed, and subjected to

ionization-time of flight (MALDI-TOF) mass spectrometry.

Fig. 3.2. Schematic of MS based amino acid analysis

HPLC-MS/MS.

3. HPLC-MS/MS: First of all, the optimized conditions for the instrumental analysis were established. Using a high-performance column and a simple isocratic elution, complete separation of the target AAs was observed. It should be noted that full baseline separation of isomeric AAs that might be indistinguishable by MS is often necessary depending on target AAs. In order to perform exact matching double ID-MS quantification, the expected AA concentrations of the hGH sample were calculated, and the same amounts of isotope labeled AAs were used as internal standards. In doing so, issues concerning measurement linearity of MS quantifications could be excluded. MS analysis was performed on a triple-quadrupole mass spectrometer using multiple reaction monitoring (MRM) mode. The ratio of peaks from unlabeled and isotopically labeled AAs was calculated, and doing so the quantity of hGH was determined.

#### **3.3.2 Analysis using isotope labeled peptides**

In 1980s, a research group reported the preparation and use of a stable isotope-incorporated peptide for measuring endogenous peptides in biological extracts (Desiderio and Kai, 1983; Desiderio et al., 1984). The pioneer work by this group seems to be the first attempt to use isotope labeled synthetic peptides as internal standards to measure the level of a specific peptide using mass spectrometry. After that, another research team described the use of this approach for the absolute protein quantification where a target protein was proteolyzed and quantified using LC-MS/MS with a stable isotope labeled peptide as an internal standard through (Barr et al., 1996). The rapid advances in mass spectrometry based proteomics have greatly improved the analytical sensitivity and made peptide based analysis possible to detect targets in a complex matrix (Anderson et al., 2004; Gerber et al., 2003; Kirkpatrick et al., 2005; Mayya et al., 2006; Putz et al., 2005; Stahl-Zeng et al., 2007). The principal of this approach is that the isotopically labeled synthetic peptide is spiked to the sample, and the mixture is subjected to enzymatic digestion followed by LC-MS/MS. Although this advanced technique is now commonly used for protein quantification, but this approach still has some issues. In addition to high costs of peptide synthesis, significant errors can be generated from sample preparation for mass spectrometry, probably from inefficient and/or inconsistent protease digestion of target proteins. The reaction conditions for protease digestion can be optimized, but the estimated values are likely to be less than the real concentrations of the target proteins. Recently, scientists from multiple NMIs collaboratively investigated the applicability of protein quantification by ID-MS using isotopically labeled synthetic peptides as standards (Arsene et al., 2008), which is briefly summarized below.


3. LC/MS: After proteolysis by trypsin, the sample containing the internal standards was subjected to LC/MS. The amounts of unlabeled and labeled peptides were monitored by revered-phase LC/ESI-quadrupole MS using certain ion traces. Comparing the signal ratio of unlabeled and labeled peptides for the standard solution of known concentration to that for the unknown sample, the quantity of hGH was calculated.
