Peptide Mass Fingerprinting PMF

8 Protein Identification Strategies 15.2.8.1 Peptide Mass Fingerprinting (PMF) 

Spectra to be analyzed via PMF are derived from a protein sample been treated with an enzyme (e.g., trypsin) or other chemical (cyanogen bromide) with specific cleavage activity. The experimental m/z values for each peptide are converted into peptide masses and compared with the theoretical mass 

PMF is generally used to identify proteins that have been previously separated by 2-D GE so that additional information including the molecular weights and isoelectric points can be used to supplement PMF identification. PMF is not well suited for searching expressed sequence tag (EST) databases that contain incomplete gene coding information for particular ESTs and it is not adequate for the analysis of complex protein mixtures in solution. 

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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). 

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. 

Fig. 3. Mascot peptide mass fingerprint (PMF) search page.

Fig. 4. Mascot search results summary page using peptide mass fingerprint (PMF) data from the spectrum in Fig. 1.

The QMS platform combines the identification of proteins with their quantitative detection in one procedure. While protein identification can be deduced from peptide mass fingerprinting (PMF) or MS/MS spectra (see section Mass Spectrometry (MS)), protein quantitation is based on analysing either peak areas or signal intensities, or a combination of both. Several computer programs, in most cases reagent specific ones, are available. For each peak, quantitation values are calculated before differentially expressed proteins are identified by the comparison of control and treated samples. 

The profile of the masses of the peptides obtained by one of these methods is compared by means of a computer with all predicted peptide digests from a database of proteins to identify the best possible matches. This is termed peptide mass fingerprinting (PMF). A protein generally can be identified using the mass of four to six of its cleavage peptides having masses in the range 700-3000 Da and determined with an accuracy of 0.05 to 0.0005 %. This is improved further if the molecular mass of the protein also is provided [83-86],... 

Figure 4.2 Schematic overview of two protein identification strategies commonly followed in proteomics. Protein samples are separated by either two-dimensional (2-D) or one-dimensional (1 -D) polyacrylamide gel electrophoresis (PAGE). In both strategic tracks, proteins are converted into a set of peptides by enzymatic digestion (e.g., with trypsin) prior to MS analysis. Peptide mass fingerprinting (PMF) by MALDl MS is predomi-...

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. 

Database searching with m/z data from peptide mass fingerprints (PMF) or uninterpreted MS-MS spectra was pioneered by several groups [4-6]. 

Peptide mass fingerprinting (PMF) of tryptic digests of both the modified and the tmmodified protein (complementary peptide mapping). By careful comparison of the two spectra, m/z shifts can be found, from which the identity of the modification may be elucidated, as well as the tryptic fragment(s) that are actrrally modified. When the amino-acid sequence of the protein is known (and vahdated), the position of the modification may be known. For example. 

Figure 7.1 The complexity of the proteins of the grape berry mesocarp of Vitis vinifera L. cv. Gamay noir. 2-DE map of proteins identified by MALDI-TOF Peptide-Mass-Fingerprint (PMF) (Reproduced from Proteomics, 2004, 4, 201-215, Sarry et al., with permission of Wiley-VCH)...

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