Peptide mass fingerprinting mapping

Mass spectrometry-based protein identification protocols have propelled proteomics to the forefront of biomedical research. Provided with available genomic and/or protein sequence information in databases, protein identification by MALDl-based peptide mass fingerprint mapping and LC-MS/MS and MALDl-TOF/TOF peptide sequencing with CAD fragmentation is efficient. However, in situations in which sufficient genome and protein sequence data are unavailable, mass spectrometric methods can be used for de novo protein sequencing. The mass measurement accuracy of MS provides a unique capability to... 

The approaches described in the previous section enable the molecular-mass determination of intact proteins, generally with an accuracy better than 0.01%. Further stractural characterization of the protein requires determination of possible post-translational modifications (PTM) as well as the amino acid sequence. In addition, issues related to tertiary and quaternary stracture of the protein, the presence of cofactors, etc., may be relevant. LC-MS plays an important role in the primaiy and secondaiy stractural characterization of proteins, i.e., in terms of amino-acid sequencing and PTM. The procedure generally involves chemical or enzymatic treatment of the intact protein, acquisition of a peptide map or peptide mass fingerprint by either direct infusion (nano-)ESI-MS or RPLC-MS, and the amino-acid sequencing of individual peptides by means of product-ion MS-MS. Further experiments may be needed in relation to PTM, as outlined in more detail in Ch. 19. 

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. 

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)...

Sagliocco, F., Guillemot, J.-C., Monti hot, C., Capdevielle, J., Perrot, M., Ferran, E., Ferrara, P., and Boucherie, H. 1996. Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting. Yeast 12,1519-1533. 

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.

Peptide-mass fingerprinting. In this approach, also known as peptide-mass mapping, the protein is first subjected to enzymatic digestion to generate a set of peptides that are unique to this protein (Section 8.4) [20,42-47]. The molecular mass of each fragment is determined accurately (within 0.5 Da) using MALDI-MS or ESI-MS. Correlation of these masses with the theoretical peptide... 

The use of intact ion masses to identify proteins is known as peptide mapping or peptide mass fingerprinting (PMF). The principle of the technique is that each protein can be uniquely identified by the masses of its constituent peptides, this unique signature being known as the peptide mass fingerprint. PMF involves the following steps ... 

Fig. 21 Schematic map of histone modifications identified in a single study by peptide mass fingerprinting [375], Each core histone is represented by a pair of horizontal lines. The color-coded bars on the top line indicate the sites of previously identified modifications, while the new sites of modification identified in this study have been added to the bottom line. Sites enclosed within brackets indicate modifications that could not be definitively localized to a specific residue. Reproduced from [375]...

Figure 5. Maldi-TOF-TOF mass analysis of CCBE. (E), peptide mapping of CCBE digested by Trypsin (F) and (G) are peptide mass fingerprint of the two main polypeptides 1764.0 and 1932.7488, respectively. (Cited from reference 142]).

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