MALDI mapping

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

Kirpekar, F. Douthwaite, S. Roepstorff, P. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry. RNA 2000, 6,296-306. 

Wall, D. B. Kachman, M. T. Gong, S. Hinderer, R. Paras, S. Misek, D. E. Hanash, S. M. Lubman, D. M. Isoelectric focusing nonporous RP HPLC A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal. Chem. 2000, 72, 1099-1111. 

It should be pointed out that FAB, MALDI, and ESI can be used to provide ions for peptide mass maps or for microsequencing and that any kind of ion analyzer can support searches based only on molecular masses. Fragment or sequence ions are provided by instruments that can both select precursor ions and record their fragmentation. Such mass spectrometers include ion traps, Fourier transform ion cyclotron resonance, tandem quadrupole, tandem magnetic sector, several configurations of time-of-flight (TOF) analyzers, and hybrid systems such as quadrupole-TOF and ion trap-TOF analyzers. 

FIGURE 9.6 The peptide and small protein map from a 100 pL human plasma injection. Columns sample preparation SCX RAM analytical column chromolith performance RP-18, 100 x 0.1 mm I.D. Minute fractions were analyzed using MALDI-TOF MS. Fraction numbers correspond to the time scale. Dot size is related to signal intensity. 

Beardsley, R.L., and Reilly, J.P. (2002) Optimization of guanidination procedures for MALDI mass mapping. Anal. Chem. 74, 1884-1890. 

Sinz, A., Kalkhof, S., and Ihling, C. (2005) Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry. /. Am. Soc. Mass Spectrom. 16(12), 1921-1931. 

Stony Brook synthesized a photoreactive benzophenone containing inhibitor (39, Fig. 14) which efficiently labeled the active site of the enzyme. The photoinhibition was prevented by adding native Ras to the reaction mixture. That competition indicated that the labeling was specific at the active site. Peptide mapping of the labeled enzyme by HPLC, Edman sequencing and MALDI-MS allowed the identification of key amino acids in the substrate binding, as Asp-110 and Asp-112 in the a-subunit [126]. 

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDI-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

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. 

There are some inherent methods to increase the reliability of a MALDI mass mapping identification. If trypsin was used as an enzyme for digestion, the sequences. . . RR.. . , . . . RK.. . , . . . KR.. . , or. . . KK.. . often produce ragged ends owing to partial cleavage behind the first lysine or arginine. This can be used to increase the confidence of an identification (Jensen et al., 1996). 

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. 

Laugesen, S. Roepstoiff, P. Combination of Two Matrices Results in Improved Performance of Maldi MS for Peptide Mass Mapping and Protdn Analysis. J. Am. Soc. 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

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). 

---