Peptide Mapping and MALDI Mass Spectrometry

An alternate method of ionization is described in the boxed essay "Peptide Mapping and MALDI Mass Spectrometry in Chapter 25. 

The Edman Degradation and Automated Sequencing of Peptides 1144 Peptide Mapping and MALDI Mass Spectrometry 1146 25.24... 

Peptide Mapping and MALDI Mass Spectrometry 1058 Oh NO It s Inorganic 1074 Chapter 26... 

Eckerskom, C., Stmpat, K., Kellermann, J., Lottspeich, F. and Hillenkamp, F. (1997) High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry. J. Protein Chem. 16, 349-362. 

Even though the MALDI peptide mass mapping technique is very powerful, it has limitations. It requires well-separated proteins, is less sensitive than identifications based on electrospray tandem mass spectrometry, can only identify proteins whose complete sequences are available in databases, and does not produce redundant information. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Figure 2. Peptide maps (A-C) and MALDI-TOF mass spectra (D-F) of PVDF-bound transferrin (53 pmol) digested with trypsin in the presence of 50 pi of 1% RTX-100/10% acetonitrile/100 mM Tris, pH 8.0 (A,D), 1% octylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (B,E), and 1% decylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (CJF) as described in Materials and Methods. Ninety percent of the digestion was analyzed by HPLC ( 29 pmol based on Table I) and 0.5% ( 150 fmol) was used for MALDI-TOF mass spectrometry. Peptides 1 and 2 in A-C were amino terminally sequenced (Table II) and analyzed by MALDI-TOF mass spectrometry (Figure 3).

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... 

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.

Biological materials often contain proteins that must be identified. Recent advances in mass spectrometry have made peptide mapping a convenient tool for this purpose. The protein in question is selectively hydrolyzed with a peptidase such as trypsin and the mixture of peptides produced is analyzed by matrix-assisted laser desorption ionization (MALDI) as illustrated in Figure 25.10. 

Traditional methods to generate peptide maps involve fractionation of complex mixtures of peptides in a protein digest either with one-dimensional SDS-PAGE or RP-HPLC [28,29]. The mass spectrometry peptide-mapping protocol, in principle, is similar to these techniques, but it provides an added dimension of structure-specific data (i.e., the molecular mass). MALDI-MS [30,31], ESl-MS [32], LC/ESI-MS [33], and CE/ESI-MS [34] have currently replaced the traditional biochemical approaches. MALDI allows the direct analysis of unfractionated protein digests. The commonly used matrices are sinapinic acid, a-cyano-4-hydroxy cinnamic acid (a-CHCA), and 2,5-dihydroxybenzoic acid (DHB). 

Figure 9 (A) Reflector MALDI mass spectrum of an in situ digest of apo-transferrin taken from the 2D map of rat sera displayed in Figure 4, which were alkylated with do-acrylamide and ds-acrylamide and mixed in a 30/70% ratio. (B) and (C) are two short intervals taken from (A), and are associated with the two indicated peptide sequences. (Reproduced from Gehanne S, Cecconi D, Carboni L, et al. (2002) Quantitative analysis of two-dimensional gel-separated proteins using isotopically marked alkylating agents and matrix-assisted laser desorption/ionization mass spectrometry. Rapid Communications in Mass Spectrometry 16 1692-1698.)...

Figure 4 Phosphopeptide identification by MALDI-TOF-MS mapping combined with alkaline phosphatase treatment. (A) The MALDI-TOF-MS spectrum of a proteolytic digest. Phospho-peptides are indicated by peaks shifted by multiples of 80 Da (HPO3 = 80Da) relative to predicted unphosphorylated peptide masses. (B) The disappearance of such peaks upon treatment with a phosphatase confirms their identity as phosphopeptides. (Reprinted with permission from McLachlin DT and Chart BT (2001) Analysis of posphorylated proteins and peptides by mass spectrometry (review). Current Opinion in Chemicai Bioiogy5 5) 591-602 Elsevier.)...

FIGURE 8.16 Three views of an unknown protein obtained by MALDI-TOF mass spectrometry (a) linearmode mass spectrum of the intact protein, (b) mass map of the tryptic peptides derived from the unknown protein, and (c) PSD mass spectrum of peptide I from the mixture in (b). (Reprinted with permission from reference I i). 

---