Mass mapping, enzymatic digests

In Chapter 4 we described a number of examples using plasma desorption mass spectrometry to map enzymatic digests. However, continuing an earlier example from this chapter, plasma desorption mass spectrometric analysis of amyloid peptides (PAi 4o and PAi 2) digested with pepsin resulted in peaks at m/z 561.1,613.1, 746.1, 1492.2, 1999.2, 2199.9, and 2315.2 that could be assigned to the peptide fragments ... 

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... 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

The other isoforms, Iso-2 and Iso-3, expressed in the E. coli fermentation of IFN-a-2b, were characterized using a similar approach. The isolated Iso-2 and Iso-3 were enzymatically digested with trypsin, and the resulted peptide mixtures were mass-mapped using RP-HPLC/ESI-MS. The results indicated that Iso-2 was a correctly folded IFN-a-2b acetylated on the amino group of the N-terminal cysteine. Iso-3 was similarly determined to be a glutathionated form (Cys-98) of the partially reduced IFN-a-2b that was pyruvated on the N-terminal cysteine. The complete structures for IFN-a-2b, Iso-2, Iso-3, and Iso-4 are shown in Figure 19-11. 

An important development in high-throughput protein identifieation is the introduction of protein database searching [111]. After separation on ID- or 2D-GE, the proteins were blotted onto a membrane and enzymatically digested after reduction and alkylation. The tryptic peptide mixture is analysed by MALDl-MS to achieve a peptide map or peptide mass fingerprint (PMF). The m/z information of the peptides is used to search the protein database, e.g., the Protein Identification Resource (PIR) database [112-114]. If the mass of just 4-6 tryptic peptides is accurately measured (between 0.1 and 0.01%), a useful database search can be performed. 

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). 

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... 

While simple molecular weight measurements do not provide information on the amino acid sequences of peptides, they have been profoundly useful for verifying sequences which are inferred from the nucleic acid sequences of the genes encoding the peptide. In particular, they have been used to verify the sequences of peptides produced by recombinant techniques or by total chemical synthesis, or to reveal possible post-translational modifications. More specific information, however, can be obtained by comparative mass mapping of tryptic (or other enzymatic) digests. This approach is particularly useful when the molecular-ion mass exceeds the mass range of the plasma desorption technique. 

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