| Abstract: | The performances of five different ESI sources coupled to a polystyrene–divinylbenzene monolithic column were compared in a series of LC‐ESI‐MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low‐flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest‐quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures – arguably due to an increased number of high intensity precursor ion candidates. |
