LC-ESMS

Table 5.9 Peptides detected during the LC-electrospray-MS (LC-ESMS) analysis of the endoproteinase Lys-C digest from native cytochrome c". Reprinted from Biochim. Biophys. Acta, 1412, Klarskov, K., Leys, D., Backers, K., Costa, H. S., Santos, H., Gnisez, Y. and Van Beenmen, J. J., Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides", 47-55, Copyright (1999), with permission from Elsevier Science...

The measured masses are obtained from the LC-ESMS analysis. 

The sequence was confirmed from a separate LC-ESMS/MS analysis. One Hg atom (Mr 200.6) is included in the calculated molecular weight. Peptide containing intra-sulfide bridge at positions 96 and 104. 

Our initial applications of the stepped collision energy LC-ESMS approach involved selective detection (and differentiation) of N- or O-glycosylated peptides (5,6), and detection of phosphorylated peptides (7,8,11). The method has been in routine use in our laboratory for over two years and during this time has become one of the mainstays of our work in characterizing protein modifications. Here we present some of our more recent studies on protein glycosylation and phosphorylation, and illustrate a preliminary evaluation of the stepped collision energy LC-ESMS method for selective detection of sulfated and acylated peptides in protein digests. 

Figure 1. Stepped collision energy LC-ESMS for gD-2 glycoprotein digested with neuraminidase/trypsin illustrating selectivity of the m/z 204 (A) and 366 (B) carbohydrate-selective markers when compared to the summed trace for m/z 400-2000 (C).

The previous experiment will detect both N-linked and 0-linked glycopeptides. These two can be distinguished by removing the N-linked oligosaccharides with PNGase F, and repeating the carbohydrate-selective LC-ESMS experiment. This time only 0-linked glycopeptides will be observed in the marker ion traces (6). 

Figure 3. Stepped collision energy scanning LC-ESMS of a 150 pmol osteopontin tryptic digest comparing the total-ion trace with that of the sum of the two phosphate-selective markers (m/z 63 and 79).

Figure 5. Stepped collision energy scanning LC-ESMS of 500 pmol 6-casein Lys-C digest spiked with the Pam3Cys showing the TIC and the summed marker-ion traces for m/z 114,239,256 and 257.

Mass Spectrometry and Other Analytical Procedures. Liquid chromatography electrospray mass spectrometry (LC-ESMS) was performed on approximately 1 pg protein samples with a Perkin Elmer Sciex API-300 triple quadrupole mass spectrometer fitted with an articulated ion spray source and set to scan over a range of 400-3000... 

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