Peptide and Protein Analysis

Table 5.5 Nomenclature of the ions formed in the mass spectral fragmentation of polypeptides. From Chapman, J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. Reproduced by permission of Humana Press, Inc. 

J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. 

F. Klink, Introduction to Protein and Peptide Analysis with Mass Spectrometry (Fullerton, CA Academy Savant, 2004), computer training Program CMSP-10. 

Fajer, P. G. (2000). Electron spin resonance spectroscopy labeling in proteins and peptides analysis. In Encyclopedia of Analytical Chemistry, (R. Meyers, ed.), pp. 5725-5761. Wiley, Chichester. 

The challenge of proteiomic and metabolomic analysis lies in the complexity (e.g., PTMs of proteins and the array of different chemical classes of metabolites), and the large range of concentrations, of the components present in the sample and in the need for high-throughput and reproducible methodologies for their identification and quantification. A detailed discussion of protein and peptide analysis by MS may be found elsewhere in this volume (see Chapter 9.12). 

Historically, most MSI experiments have focused on protein and peptide analysis. However, protein analysis still suffers from low sensitivity, ion suppression issues, and difficulties in identification. This can be partially overcome by performing on-tissue enzymatic digestion with specific enzymes, usually trypsin, to generate peptides. Peptides have higher ionization efficiency and high-resolution measurements or MS/MS experiments can be performed for accurate identification. The drawback is that important information, such as posttranslational modifications, is lost (3). 

The hybrid quadrupole time of flight mass spectrometer (QTOF) was introduced as a mass spectrometer capable of tandem MS with particular emphasis on its applicability for protein and peptide analysis. It combines the simplicity of a quadmpole MS with the high efficiency of a... 

Protein and Peptide Analysis by Mass Spectrometry, edited by John R. Chapman, 1996 60. Protein NMR Protocols, edited by David G. Reid, 1997 59. Protein Purification Protocols, edited by Shawn Doonan, 1996... 

Liu, J., and Lee, M.L. Permanent surface modification of polymeric capillary electrophoresis microchips for protein and peptide analysis. Electrophoresis, 27, 3533, 2006. 

Astorga-Wells J et al (2005) Microfluidic systems and proteomics applications of the electrocapture technology to protein and peptide analysis. Anal Biochem 345(1) 10-17... 

The essential feature of the electrospray spectrum that enables compounds of high mass to be examined is the multiple charging. Typical mIz values are in the region below 5kDa and, consequently, typical quadrupole or related instruments can easily analyze the ions with no modification being needed. As the method accepts a flowing solvent, it is ideally coupled to liquid chromatographic columns and has major applications in the field of protein and peptide analysis. 

Sample Preparation for Protein and Peptide Analysis by MALDI-MS... 

Figure 5.3 Protein loading capacity of RP-HPLC materials of different particle sizes. Source Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.

Rodriguez, I. Li, S.F.Y. Surface deactivation in protein and peptide analysis by capillary electrophoresis. Anal. Chim. Acta 1999, 383, 1. 

FIGURE 10.1 Molecular weight strategies for protein and peptide analysis by time-of-flight mass spectrometry. 

With respect to resolving power (theoretical number of plates) and separation time, no doubt, capillary electrophoresis (CE) is the ultimate separation technique for complex peptide samples, and its combination with ESI MS online as well as MALDI MS off-line has been demonstrated many times [222-229]. The main reason why CE-MS, in contrast to nano-LC-MS, has not become a widespread method for protein and peptide analysis is the maximum total sample volume that can be separated by CE. In contrast to nano-LC, where many himdred microhters of dilute sample can be loaded without compromising separation power, the performance of CE directly depends on the sample volume and works best if only 50 nL or less is loaded. Recently, however, it has been reahzed that this requirement of CE is perfectly matched by nano-LC, which provides efficient sample concentration, and that the two techniques can be combined online upfront ESI or MALDI MS. For this purpose, a microfluidic chip was developed that enables, on demand, on-hne transfer (loading) of nano-LC fractions to an orthogonal CE separation channel, the effluent of which is either analyzed online by ESI MS or off-line by MALDI MS [230-232]. 

Analytes are very frequently observed as protonated molecules in MALDI. This is particularly so in biological applications such as protein and peptide analysis. If approach to local thermal equilibrium is extensive, we expect the spectrum to be defined by the gas-phase proton affinities (PAs) of matrix and analyte. Consider simple biomolecular analytes such as amino acids, which have PAs from 885 kJ/mol for glycine to 1025kJ/mol for arginine.Comparing with matrix PAs, proton transfer from typical matrices to the least basic amino acid, glycine, is found to be weakly exothermic to endothermic, while more basic arginine can easily abstract a proton from the common matrices. 

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