Peptide mass mapping measurement

Fig. 6. Protein identification using a peptide map measured with a matrix-assisted laser desorption time-of-flight mass spectrometer. All the peptide extracted from the gel is measured and the set of masses is used in the database search. The mass resolution is in the order of 10,000. Individual isotopes of a 2.5 kDa peptide are clearly resolved.

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

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. 

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.

Figure 5. Mapping of protein polymorphisms by mass spectrometry. A Major Urinary Protein from Mus spretus was purified and Lys-C digestion fragments were mass measured by MALDl-ToF mass spectrometry. Four of the peptides had the same masses as the equivalent protein from Mus domesticus. However, for two of the remainder, of novel mass, tandem mass spectrometry led to the identification of an amino acid change that was consistent with the new peptide mass. (J is used to define leucine or isoleucine, which cannot be distinguished by this type of mass spectrometry).

While simple molecular weight measurements do not provide information on the amino acid sequences of peptides, they have been profoundly useful for verifying sequences which are inferred from the nucleic acid sequences of the genes encoding the peptide. In particular, they have been used to verify the sequences of peptides produced by recombinant techniques or by total chemical synthesis, or to reveal possible post-translational modifications. More specific information, however, can be obtained by comparative mass mapping of tryptic (or other enzymatic) digests. This approach is particularly useful when the molecular-ion mass exceeds the mass range of the plasma desorption technique. 

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... 

Methods to measure the structure of biopharmaceuticals include tryptic peptide mapping, HPLC, capillary electrophoresis (CE), mass spectroscopy, and circular dichroism spectra. 

Mass spectrometry is a powerful qualitative and quantitative analytical tool that is used to assess the molecular mass and primary amino acid sequence of peptides and proteins. Technical advancements in mass spectrometry have resulted in the development of matrix-assisted laser desorption/ion-ization (MALDI) and electrospray ionization techniques that allow sequencing and mass determination of picomole quantities of proteins with masses greater than 100kDa (see Chapter 7). A time-of flight mass spectrometer is used to detect the small quantities of ions that are produced by MALDI. In this type of spectrometer, ions are accelerated in an electrical field and allowed to drift to a detector. The mass of the ion is calculated from the time it takes to reach the detector. To measure the masses of proteins in a mixture or to produce a peptide map of a proteolytic digest, from 0.5 to 2.0 p.L of sample is dried on the tip of tlie sample probe, which is then introduced into tire spectrometer for analysis. With this technique, proteins located on the surfaces of cells are selectively ionized and analyzed. 

Alternatively, imaging mass spectrometry (IMS) using matrix-assisted laser des-orption/ionization (MALDI) can be used to simultaneously map the distribution of pharmaceuticals in thin tissue sections to determine how a drug is distributed in animal tissues [6-9], MALDI-IMS has been extensively employed to measure macromolecules such as peptides and proteins in tissue sections [10-13] (Figure 11.1). Although MALDI-IMS has been applied almost exclusively as an analytical tool for... 

The C-terminal sequence of P40 was confirmed by sequencing the C-terminal peptide identified from the peptide map the peak annotated LI8 on the chromatogram corresponds to the C-terminus amino acids 337-344 (measured mass 870,61 Da theoretical mass 870,96 Da). The sequence deduced from the sequencing analysis, EVVTQPQA, was in accordance with the theoretical amino acid sequence. 

Comparative Peptide Mapping This procedure is used when the protein is cleaved with specific cleaving agents and the sequence of the protein is known. A simple molecular mass measurement of the peptides in a protein digest will identify the position of a disulfide bond in the protein. 

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