**2.3 Apparatus and operating conditions**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

subjected to gas-phase fragmentation [47].

*(D) is the deamidated form of Asn (N).*

corresponding reference standard [13, 48].

**2. Experimental**

**Figure 2.**

concentration.

**2.2 Proteolysis**

**2.1 Sample preparation**

for protein identification is the top down approach, where intact molecule ions are

*MALDI in source decay analysis of a suspected illegal somatropin sample. The blue marked amino acid asp* 

Proteins with posttranslational modifications, such as glycosylation, present additional challenges since the masses of the modified peptides are different and thus do not contribute to the identification. In such cases, the protein can be analyzed by capillary electrophoresis (CE), in order to explore the heterogeneity of the protein followed by comparison of its electropherogram with that of the

MALDI-TOF-MS is very tolerant to salts and sample matrices, hence it is seldom

necessary to desalt the sample. However, sometimes it is necessary to use a C18 micro-column in order to fractionate a complex sample or enhance the target analyte

The sample to be analyzed is mixed with a matrix solution (1:1, v:v), e.g. sinapinic acid (SA) or alpha-cyano-4-hydroxycinnamic acid (ACHCA). One μl of the mixture is deposited on the MALDI target plate and allowed to air-dry (i.e., the dried-droplet method) before being placed in the mass spectrometer [19, 49].

The analyte to be digested is dissolved in ammonium bicarbonate (50 mM, pH 7.9). The intact sample is directly analyzed by MALDI in order to determine the molecular mass of the analyte. Then, 200 μl of the solution is digested by addition of 2–10 μl trypsin (200 μg/ml in 10 mM HCl). The reaction is carried out at room temperature or at 37°C for 30 minutes up to 24 hours, depending on peptide or protein in question. It has been found that 30 minutes digestion of somatropin at room temperature generated enough tryptic fragments for the MALDI analyses [50]. For more complex proteins, such as human chorionic gonadotropin, the required time

**30**

MALDI-TOF analyses are performed using either an Autoflex or an Autoflex Max (Bruker Daltonics, Bremen, Germany) reflector type time-of-flight mass spectrometer, equipped with a pulsed nitrogen laser working at 337 nm and a smartbeam II laser working at 355 nm, respectively. The Autoflex instrument is operated in the positive ion mode with delayed extraction at an accelerating voltage of 20 kV and a variable voltage reflectron. The parameter settings are optimized to analyze peptides in reflectron mode. Before analysis, the instrument is externally calibrated with Bruker Daltonics standard peptide or protein mixtures. Peptide mass peaks occurring due to autolysis of trypsin (porcine) such as 842.51 and 2211.10 Da are also used for internal calibration. Mass spectra are obtained by averaging 250 laser shots (5× 50 shots) at different positions on the sample surface. All samples being used for post source decay (PSD) analysis are analyzed in the reflectron mode. The autoflex Max instrument TOF/TOF (2 kHz MS and 200 Hz MS/MS) operates in the positive ion mode. Metastable fragmentation is induced by laser (355 nm) without the further use of collision gas. The lyophilized samples are dissolved in 300 μL ammonium bicarbonate buffer (50 mM, pH 7.5). The liquid samples are diluted with same buffer. The wells of MALDI plate are spotted with 1 μl sample/matrix solution (1:1, v:v) and allowed to air dry before being placed in the mass spectrometer. ACHCA is used for analysis of peptides. About 20 mg of ACHCA is mixed in 1 ml of ethanol: acetonitrile (ACN) (1: 1 v/v) and 0.1% trifluoroacetic acid (TFA). SA is used for protein analysis. Two different solutions of SA in water and ethanol are made as follows: 1 - Saturated solution of SA in ethanol and 0.1% TFA; 2 - Saturated solution of SA in 50% acetonitrile (ACN) and 0.1% TFA. Solution 1 is first applied on the MALDI plate on which the sample mixed with SA in 50% ACN and 0.1% TFA (1: 1) is then applied.
