Peptide digest mapping

This procedure is called Peptide Mass Mapping (PMM), which is defined as a means of protein identification by comparing observed masses (m/z values) with predicted masses of digested proteins contained in a database. 

Peterson DS, Rohr T, Svec FK, Frechet JMJ (2003) Dual-function microanalyt-ical device by in situ photolithographic grafting of porous polymer monolith integrating solid-phase extraction and enzymatic digestion for peptide mass mapping. Anal Chem 75 5328... 

An ACE-MS hyphenation was utilized for the linear epitope mapping (17). The tryptic digest of /8-endorphin was mixed with an anti-/8-endorphin antibody and subsequently analyzed by ACE-ESI-MS. The procedure requires only femtomole amounts of antibody and peptide digest. More technical details of the method are described in Chapter 13 of this book. 

To obtain optimum results in a FAB analysis samples should be relatively pure, though simple mixtures can be tolerated. We routinely perform mixture analysis with FAB, especially in our peptide mapping studies. However, certain shortcomings may arise. Complex mixtures are often plagued by a signal suppression effect where certain mixture components are preferentially ionized at the expense of others. When peptide digests are analyzed the hydrophobic components are ionized with much greater efficiency and the more hydrophilic components are suppressed. Surfactant type... 

N-alkyl silica-based stationary phases typically employed in HPLC separation of peptides and proteins have been investigated to a large extent in CEC mode. In most cases, gradient elution was required to optimize the resolution and analysis time. Tryptic digests maps of cytochrome c by pressurized CEC were better resolved than by pHPLC [93], The experiments were conducted at low pH in the presence of trifluoroacetic acid (TFA) to prevent tailing of basic peptides, on a column packed with 1,5-pm C18 silica particles. It has been... 

FIG. 5. Tryptic peptide maps of the maize-branching enzymes showing the similarity between the pair BEIIa/BEIIb and differences between the pair and BEI. Approximately 1.25 nmol of peptide digest was chromatographed on a reverse-phase column. Figure reprinted with permission from Singh and Preiss (1985). 

The peptide mass mapping strategy using trypsin and V8 protease was applied to solve structural identification problems of the variants. The comparison of the trypsin and V8 protease digest of the native GM-CSF (VO) and... 

Figure 19-25. Peptide mass mapping of in-gel trypsin digested 31-kDa band by MALDI-MS. (Reprinted from reference 138, with permission of Elsevier Science B. V.)...

Peterson, D.S. Rohr, T. Svec, F. Frechet, J.M.J. Dual-function Microanalyti-cal Device by In Situ Photolithographic Grafting of Porous Polymer Monolith Integrating Solid-Phase Extraction and Enzymatic Digestion for Peptide Mass Mapping, Anal. Chem. 75, 5328-5335 (2003). 

Figure 3. Identification of a protein by peptide mass fingerprinting. The protein constituents of pig saiiva were separated by SD-PAGE and a protein band was digested with trypsin. The resuitant tryptic peptides were mass-measured using MALDI-ToF mass spectrometry. The peptides in the mass spectrum were either derived from trypsin self-digestion (T) or were derived from the protein in the gel- Database searching with the masses of these peptides led to an unequivocal identification of the protein as SAL (salivary lipocalin). The inset map shows the theoretical tryptic digestion map of this protein, and underneath are the peptides that were observed. In many instances, smaller peptides were visible as partial digestion products.

Figure 6.4 XRCC4 composite peptide sequence map, arranged according to domain type [36], Peptides obtained using pepsin digestion at four different enzyme-to-substrate ratios (65 1 to 520 1, upper bars) and using nepenthesin digestion at four different enzyme-to-substrate ratios (0.0075 1 to 0.38 1, lower bars). This research was originally published in Ref [36]. the American Society for Biochemistry and Molecular Biology. (See insert for color representation of the figure.)...

Peptide-mass fingerprinting. In this approach, also known as peptide-mass mapping, the protein is first subjected to enzymatic digestion to generate a set of peptides that are unique to this protein (Section 8.4) [20,42-47]. The molecular mass of each fragment is determined accurately (within 0.5 Da) using MALDI-MS or ESI-MS. Correlation of these masses with the theoretical peptide... 

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