LC-MALDI MS

Chen, H.S., Rejtar, T., Andreev, V., Moskovets, E., Karger, B.L. (2005). High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis. Anal. Chem. 77, 2323-2331. 

Reference 5. With the advent of commercially available MALDI-TOF-TOF instruments the combination of off-line one- or two-dimensional LC-MALDI-MS/MS has become a popular alternative or rather a complement to LC-ESI-MS/MS in the proteomics community. 

Hofmann, S., Gluckmaim, M., Kausche, S., Schmidt, A., Corvey, C., Lichtenfels, R., Huber, C., Albrecht, C., Karas, M., and Herr, W., Rapid and sensitive identification of major histocompatibility complex class 1-associated tumor peptides by nano-LC MALDI MS/MS, Molecular and Cellular Proteomics 4(12), 1888-1897, 2005. 

The complementarity of all the data obtained in this strategy (LC-MALDI-MS and nanoLC-MS/MS) allowed to increase significantly the coverage percentage for all the gel slices. Figure 3 summarizes this strategy and technical details are presented below. 

LC-MALDI-MS experiments are also reported. The combination of both ESI and MALDI experiments shows well the advantage in term of coverage of this double ionization process. Indeed, some peptides are identified specifically from the ESI ionization process and other from the MALDI process, increasing the whole number of identified peptides. 

Moreover, we have determined the false positive rate for this approach. Many tryptic peptides originated from different proteins can be attributed to a single mass (e.g. HQHPLQCVMEK 1364.63 Da and EADFINCVIWR 1364.65 Da AM < 20 ppm). Thereby false positive identification may occur. To evaluate the false positive rate, we have selected three common proteins which were not identified during the nanoLC-MS/MS analysis. These proteins (tubulin, actin and myosin) were digested in-silico, and the generated mass lists were compared to the LC-MALDI-MS peaklist. A total of only five masses were attributed to the three... 

Table 1. Sununary of the proteins identified by nanoLC-MS/MS from a single 1D gel slice, and the increase of the coverage using LC-MALDI-MS strategy...

To conclude, we have developed a strategy which combines LC-MALDI-MS and nanoLC-MS/MS in order to identify proteins originating from ID gel with a high coverage compatible with plasma membrane study (CD98, CD71, CD44). 

To achieve the best performance in protein identification or quantitation, the extent of protein or peptide separation should match the capabilities of the technique applied in the identification or quantitation step. With LC-MALDI MS and MS/MS as analysis technique, the number of good-quality MS/MS spectra, which can be acquired from one sample spot (LC-fraction) represents one limitation the number of components in one fraction should therefore not exceed this maximum. By increasing matrix concentration more laser shots could be acquired from one spot, but analyte concentration in the crystals and detection sensitivity would... 

Compared to yeast with about 5800 ORFs which code for proteins, samples from higher organisms can be much more complex and the separation scheme has to be adjusted accordingly. For very simple species as the other extreme, already subcellular fractionation can provide the appropriate pre-separation. For example only one LC-MALDI MS/MS run of a peptide mixture derived from the separated E-coli 50S ribosomal subunit allowed to identify 30 of the 33 expected proteins (Mirgorodskaya et al. 2005a). 

For non-quantitative LC-MALDI applications, derivatization chemistry is not restricted to compounds which allow a convenient incorporation of heavy isotopes. For example for improved MS/MS detection sensitivity, tryptic peptides were labeled with sulfonated coumarin-tags at the N-termini after guanidylation of lysines (Pashkova et al. 2005). Despite reduced MS sensitivity for arginine-terminated peptides (in alpha cyano-4-hydroxycinnamic acid matrix), formation of y-ions was enhanced in MS/MS by the second mobile proton provided from the sulfonic acid group. For a SCX fraction from a E.coli hydrolysate 50% more peptides and 30% more proteins could be identified by multiplexed LC-MALDI MS and MS/MS after derivatization. 

LC-MALDI MS- and MSMS runs 22067 MSMS spectra (mean 340/run)... 

Ji, C., Li, L., Gebre, M., Pasdar, M. and Li, L. (2005) Identification and quantitation of differentially expressed proteins in E-cadherin deficient SCC9 cells and SCC9 transfectants expressing E-cadherin by dimethyl isotope labeling, LC-MALDI MS and MS/MS. J. Proteome Res. 4, 1419-1426. 

The currently most frequently applied method for LC-MALDI-MS is automated post-column fractionation and on-plate collection in discrete spots of the LC colunm effluent. After the solvent is evaporated, the matrix solution can be added, and MALDI-MS analysis of the various spots can be performed. The procedure requires a liquid-handling robot, capable of disposition of effluent fractions at discrete spots on the MALDI target. A number of ways were proposed for deposition in discrete spots on the MALDI target, e.g., blotting via direct contact between droplet and target [139-140], piezoelectric flow-through microdispensing [141], pulsed electrical-mediated droplet deposition [142], and a heated droplet interface [143], Commercial LC-MALDI-MS devices were recently reviewed [144],... 

The complementary nature of MALDI-MS and ESI-MS-MS can also be exploited to increase the proteome coverage via PSA [36]. A fraction (20%) of the column effluent of a nano-LC column was sent to ESI-MS-MS, while the rest was fractionated onto MALDI spots for automated off-line LC-MALDI-MS and MS-MS analysis. About 63% overlap in protein identified from a digest of mammalian mitochondrial ribosomes was observed, but unique proteins were also identified by either technique. 

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