Protein Analysis by MALDI-MS

The success story of MALDI started with MALDI-TOF-MS on intact proteins. Hillenkamp and Karas showed that MALDI is unique in that it can combine the abihty for i) desorbing ions of biomacromolecule into the gas phase, ii) preserving their structural integrity, and iif) being perfectly compatible with mass analyzers with almost unlimited m/z range and extraordinary sensitivity (Figs. 11.1 and 11.4) [11,13,14]. 

The analysis of intact proteins is often the first step in a series of analytical procedures revealing characteristics of cells, physiological pathways, diseases, or o-ther aspects of interest in a biological or biomedical context. Often, the proteins are separated prior to MALDI-MS, e.g., by 2D gel electrophoresis. MALDI experiments must be carried out with highly purified proteins or mixtures containing only a limited number of proteins. In cases where the full mass range of proteins is needed, MALDI-TOF-MS would be the preferred choice. 

Commonly, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) [85] and 2,5-dihydroxybenzoic acid (DHB) [17,50] are suitable for MALDI of pro- 

Example In food analysis of fish and seafood products, differentiating between individual species of pathogenic and food-spoilage microorganisms and their rapid identification are particularly inportant.. MALDI-TOF-MS protein 

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In this section, some of the general sample preparation issues that influence protein analysis by MALDI-MS experiments are described. 

Teramoto K, Sato H, Sun L, Torimura M, Tao H. A simple intact protein analysis by MALDI-MS for characterization of ribosomal proteins of two genome-sequenced lactic acid bacteria and verification of their amino acid sequences. J Proteome Res. 2007 6(10) 3899-907. doi 10.1021/pr0702181. 

The present emphasis is put on general chemistry testing and immtmoassays based on absorption, agglutination, or fluorescence detection. Also the preparation of protein samples for subsequent analysis by MALDI-MS has been... 

Sample Preparation for Protein and Peptide Analysis by MALDI-MS... 

Strategies for Using MALDI-MS in Protein Biochemistry 111 Table 3.2 Matrices and sample preparation methods for peptide analysis by MALDI-MS. 

Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis. 

Encouraged by this spectral reproducibility, we focused our efforts on the particularly challenging problem of distinguishing bacterial strains by MALDI MS. We developed a modified correlation approach22 that relies on two fundamental qualities of bacterial mass spectra. First, because different bacterial strains of the same species have substantial, if not complete, genetic overlap, most of the protein masses observed with two different strains will be identical. This feature limits the value of the biomarker approach that is commonly used to differentiate bacteria species. Second, as just noted, closely controlled sample preparation and mass analysis procedures can result in highly reproducible results.22 The modified correlation approach takes advantage of subtle, yet reproducible, differences in mass spectra obtained from dif-... 

Inspection of the UV chromatogram from the LC-ESI MS analysis of the tryptic digest of the protein revealed a peak not present in the digestion of wild-type TTR, at retention time 25.95 min. The [M + H]+ of this variant peptide (m/z 1392.6) was consistent with a 27 Da increment to the normal tryptic peptide T4, 22GSPAINVAVHVFR34. The location of the mutation was confirmed by MALDI MS analysis of the chromatographic fraction collected between retention times 25.0-27.5 min. In the MALDI mass spectrum, peaks at m/z 1365.2 (wild-type T4) and m/z 1392.1 (variant T4) are both present (Fig. 8). This observation confirmed that the mass difference between the variant and the wild type peptides is indeed +27 Da. The only possible amino acid substitution that would give rise to a +27 Da shift in that peptide is Ser23 —> Asn. 

Capillary isoelectric focusing can be applied as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) [69,70]. The exact timing of the collector steps in the interface is based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. During the collection a sheath flow fraction collector is used to maintain the permanent electric current. 

Pesavento, I.C., Bertazzo, A., Flamini, R., Dalla Vedova, A., De Rosso, M., Seraglia, R. and Traldi, P. (2008) Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins,/. Mass Spectr., 43(2), 234-241. 

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