NanoLC-MALDI

In order to meaningfully compare MALDI detection limits to the other techniques, specifically defined analytical conditions must be established. The case of peptide mapping is a good starting point because this class of chemical species ionizes well using both MALDI and ESI. Moreover, a relatively large body of work using both NanoLC/ESI and NanoLC/MALDI has been reported from which some initial conclusions may be drawn. As... 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Use these solutions directly for MALDI-MS and nanoLC-ESI-MS or desalt them... 

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. 

Figure 5. Summary of the strategy applied for a better sequence coverage of the identified proteins in ID gel. The first step is the analysis of the raw peptide mixture by MALDI. The step 2 corresponds to the nanoLC-MS/MS analysis of the peptide digests, and the step 3 corresponds to the off-line fractionation of the peptide digest and MALDI-MS analysis. The combination of these approaches allow a better coverage of the different proteins identified.

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...

Accession number Protein Peptides identified by nanoLC-MS/MS Coverage (%) Peptides identified by LC-MALDl Combination MALDI/ESl ... 

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). 

As highlighted in the reviews from Li et al. [71] and Hummon et al. [41], in addition to the mollusk single-cell models, MALDI-MS associated or not to nanoLC and on-line LC-ESI-MS/MS has been successfully applied to a wide variety of animal species from invertebrates to vertebrates. 

Williams, D.K. Hawkiidge, A.M. Muddiman, D.C. Sub-ppm mass measurement accuracy of intact proteins and product ions achieved using a dual electrospray ionisation quadrupole FT-ICR mass spectrometer. J. Am. Soc. Mass Spectrom. 2007,18 ), 1-7. Witt, M. Fuchser, J. Baykut, G. FT-ICR mass spectrometry with NanoLC/microelec-trospray ionization and MALDI Analytical performance in peptide mass fingerprint analysis. /. Am. Soc. Mass Spectrom. 2003,14(6), 553-561. 

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