Proteins by peptide mass fingerprinting

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

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. 

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 availability of the sequence of two genomes greatly facilitated the identification of proteins by peptide mass fingerprinting. Therefore we started to establish a public database of the sequenced H. pylori strain 26695 where our 2-DE gel patterns and results of protein species... 

Pappin, D., et al. (1993). Rapid Identification of Proteins by Peptide-mass Fingerprinting, Current Biology 3 327-332. 

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.

Arnott, D. P Henzel, W. J. Stults, J. T. 1995. Identification of proteins from two-dimensional electrophoresis gels by peptide mass fingerprinting. In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, Snyder, A. E, ed., Washington, D.C. American Chemical Society, 619,226-243. 

Cottrell JS. Protein identification by peptide mass fingerprinting. Pept Res 1994 7 115-124. 

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). 

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