Adaptation of LC-MALDI MS workflow to sample complexity

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 

Selection of parent ions for MSMS after completion of MS data acquisition 

Acquisition of MSMS specto for all selected preci sor ions from whole plate (3 h for about 500 candidates) 

Database search of all MSMS spectra (peptides in different spots fitting the same protein are scored together) 

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). 

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