Peptide mass fingerprinting

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

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)... 

Stuhler K, Meyer HE. 2004. MALDI more than peptide mass fingerprints. Curr Opin Mol Ther 6 239. 

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)... 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

Zabet-Moghaddam, M. et al., Pyridinium-based ionic liquid matrices can improve the identification of proteins by peptide mass-fingerprint analysis with matrix-assisted laser desorption/ionization mass spectrometry. Anal. Bioanal. Chem., 384, 215, 2006. 

Characterization of noncovalent bonding of the proteins can also be done using MS. For example MALDI MS has been used in measurement of the molecular mass of the noncovalendy linked tetramer of glucose isomerase, a complex consisting of identical monomers of 43.1 kDa each. MALDI-TOFF peptide mass fingerprinting combined with electrospray tandem mass spectrometry can efficiently solve many complicated peptide protein analysis problems. 

Fig. 17.3. The infected bile-ome Two-dimensional electrophoresis gel of F. hepatica infected host bile. Run on a 17 cm, pH 3-10, IPG strip in the first dimension and then on 12.5% SDS-PAGE gel in the second dimension with Coomassie blue staining. Host and parasite proteins were identified via their peptide mass fingerprints (Morphew, 2004, unpublished). Valid parasite secreted proteins can only come from in vivo proteomics.

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