Peptides routine spectrometry

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

If the scope of mass spectrometry is limitless, why are the applications of clinical MS almost completely small molecules The answer is that most clinical tests analyze small molecules, biomarkers that are either metabolites or steroids and, hence, mass spectrometers would target those first. Perhaps a more complete answer would also include that methods must be very robust, easily reproduced in different labs, reliable, and subjected to an extensive array of validation tests. Although peptide and protein analysis is increasing rapidly in clinical labs, the MS approaches to these assays is lagging behind somewhat. MS techniques targeting these peptides and proteins exist, but they are primarily in the research stage, with few systems and methods subjected to the clinical rigors of validation. Once the necessary validations occur and methods simplified, it will only be a short time before MS is used routinely in clinical proteomics. 

Mass spectrometry provides a more direct and precise technique to study histone modifications. As with the other methods discussed above, mass spectrometry also has several pitfalls that should be taken into account when analyzing histone modifications. First of all histones and especially the core histones H3 and H4 are rich in lysine residues. Consequently, trypsin as an enzyme that is routinely used for the identification of proteins via peptide mass fingerprints cannot be used for regular in gel digestion of histones. Other enzymes that have a different specificity (such as Asp-N or Arg-C) are more frequently used in the analysis of histones [25]. A drawback... 

Liquid chromatography coupled to tandem mass spectrometry (LC-MS) is a powerful technique for the analysis of peptides and proteins. While numerous methods for coupling MS to LC have been explored and used to analyze copious samples (as seen in Table 3.3), it is ESI that has transformed LC-MS into a routine procedure sensitive enough to analyze peptides and... 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

The coupling of HPLC with NMR represents a powerful method for the high-throughput screening of peptides in mixtures run in stop-flow and continuous-flow modes. It is possible to obtain routine high-quality HPLC/NMR ID NMR data with as little as 5 pg of compound in a chromatogram peak. However, the HPLC/NMR technique cannot be favorably compared to mass spectrometry techniques (HPLC/MS) in terms of sensitivity and speed of analysis. To date, the majority of reports of the use of HPLC/NMR have been for drug metabolites.1 ... 

Of the 20 respondents, 19 use mass spectrometry for routine analysis of peptides, 16 use HPLC, and seven use amino add analysis. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

The advent of FAB mass spectrometry has allowed the routine molecular weight determination of polar molecules, without derivatization, up to ca 3,000 Daltons, and in exceptional cases, within 1 mass unit to the region of 8,000 Daltons. This advance, coupled with FAB fragmentation, and enzymic digestion techniques, has allowed the rapid solution of a number of problems in protein and peptide chemistry - problems which were hitherto rather difficult to solve. Examples are given. 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

The development and use of various protein sequence databases for automated search routines (Eng et al., 1994 Clauser et al., 1999) are an essential component of protein analysis that uses mass spectrometry techniques. These programs (i.e., SEQUEST, MASCOT) require only a few peptides for matching therefore, the absence of a match for a particular peptide does not affect the search performance. Using protein database searches provides an efficient way of confirming a putative sequence from corresponding full-scan mass spectrometry and MS/MS data. 

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