Sample protein/peptide analysis

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

Figure 12.5 illustrates a typical problem of analysis of minor components present in a matrix of highly abundant ones. Despite the availability of large LC-MS peak capacity (Table 12.3), the number of peptides detected in a semm/plasma digest does not exceed several hundreds (Kapp et al., 2005). These peptides typically match 30-50 high abundant proteins. We believe that the maj ority of remaining proteins/peptides in the sample are present at concentrations well below the LOD of MS instrument. 

Janini, G.M., Zhou, M., Yu, L.-R., Blonder, J., Gignac, M., Comads, T.P., Issaq, H.J., Veenstra, T.D. (2003). On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry evaluation for peptide analysis and protein identification. Anal. Chem. 75, 5984—5993. 

The application of FAB to peptide analysis has two general advantages it typically generates accurate molecular weight information due to the mass analyzers to which it is interfaced, and it offers rapid analysis. Given that FAB is not commonly applied to the analysis of proteins due to its limited mass range, routinely of the order of 5000 to 8000 Da, and relatively poor sensitivity (typically requiring >500 pmol), we will only briefly focus on the sample preparation and analysis procedures for peptides. 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

FIGURE 7.44 MALDI mass spectra from on-CD analysis of the phosphopeptide-con-taining sample, (a) Peptide mass spectrum after concentration/desalting. A database search showed that the sample contained bovine protein disulfide isomerase. (b) Phosphopeptide enrichment by IMAC. Two phosphopeptides at m/z 964 and 2027 were recognized (c) Phosphopeptide enrichment followed by enzymatic on-column dephosphorylation using alkaline phosphatase. Two phosphopeptides at m/z 884 and 1947 were recognized from the mass shifts of 80 Da from (B), which were resulted from dephosphorylation [794]. Reprinted with permission from the American Chemical Society. 

Removal of the precipitated serum proteins and analysis of the proteolytic degradation of the injected peptide in the different plasma samples are done in accordance with steps 3-6 in Subheading 3.2. 

In recent years there has been a growing interest in the use of electrospray ionization-mass spectrometry (ESI-MS) either as a stand-alone technique, or following an analytical separation step like CE, to study and measure a wide variety of compounds in complex samples such us foods (Simo et al. 2005). ESI provides an effective means for ionising from large (e.g., proteins, peptides, carbohydrates) to small (e.g., amino acids, amines) analytes directly from solution prior to their MS analysis without a previous derivatization step. Santos et al. (2004) proposed the use of CE-ESI-MS for the separation and quantification of nine biogenic amines in white and red wines. More recently, the possibilities of two different CE-MS set-ups, namely, capillary electrophoresis-electrospray-ion trap mass spectrometry (CE-IT-MS) and capillary electrophoresis-electrospray-time of flight mass spectrometry (CE-TOE-MS) to analyze directly biogenic amines in wine samples without any previous treatment has been studied (Simo et al. 2008). 

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