Protein mass fingerprinting

The sensitive requirements for exposure assessment have been met by the recent development of mass spectrometry (MS) techniques for the characterisation of proteins expressed by a genome, tissue or cell. Of particular interest is the matrix-assisted laser-desorption/ionisation (MALDI), which makes it possible to obtain protein mass fingerprinting for a wide range of proteins by MS. This method involves selectively cutting proteins by enzymatic actions, and comparing the fragment masses with theoretical peptides available in bio-informatic databases. 

Penicillin Binding Protein Pentasaccharide Peptide Mass Fingerprint Peptide YY Peptidoglycans Peptidyl Transferase Center Peptidyl-Dipeptidase PERI... 

Variations on the spectral peaks from different species of the same genus were also observed. Three species of Pseudomonas produced the spectra shown in Figure 14.2. These spectra are clearly unique and were used to correctly identify unknown samples. Because of peak ratio reproducibility issues in bacterial protein profiles obtained by MALDI MS,11 a fingerprint approach that had been used for other mass spectrometry approaches has not been used. The profile reproducibility problem was first recognized by Reilly et al.12,13 and later researched by others in the field.14,15 As a later alternative, a direct comparison of the mass-to-charge ratio (m/z) of the unknown mass spectral peaks with a database of known protein masses has been used to identify unknown samples.14... 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

PAPPIN, D.J.C., HOJRUP, P., BLEASBY, A.J., Rapid identification of proteins by peptide-mass fingerprinting, Curr. Biol., 1993,3, 327-332. 

Protein Identification Strategies 15.2.8.1 Peptide Mass Fingerprinting (PMF)... 

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-... 

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... 

MULTIIDENT (Proteins Identification Using pi, MW, Amino Acid Composition, Sequence Tag and Peptide Mass Fingerprinting Data)... 

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