Abstract
The LTQ Orbitrap Velos offers many modes of operation for analyzing peptides in proteomic studies. Due to the time-dependent nature of LC-MS/MS analyses choice of chromatographic conditions has direct implications on the number of peptides identified from complex protein digests. In this study we examined the effects of LC gradient time, column particle size, and fragmentation mode on both the number of protein identifications and associated peptide scores with a standard Ecoli tryptic digest. Using the EASY-nLC (Proxeon) combined with manually pulled and packed-tip fused silica columns, different C18 particle sizes and LC gradient conditions were investigated. This was done acquiring fragment ion data at both nominal mass (CID LTQ) and at high mass accuracy (CID/HCD Orbitrap). As expected the number of peptide identifications increased with increased gradient elution time, for example, the number of peptides tripled by extending the elution gradient from 1 to 4 hours. The use of smaller particle size (3um vs. 5um C18) also showed an improvement in the number of identifications on average by 5-10%. We also evaluated the effect of mass accuracy of the fragment ions on the Mascot ion scores from the database search result. Interestingly, Mascot scores of the same peptide m/z generated via CID and scanned in the LTQ as compared to either CID or HCD scanned in the Orbitrap tend to show higher scores for more peptides when the length is 15 residues or greater. By contrast, when peptides were 10 residues or less the higher mass accuracies afforded by the Orbitrap tended to show more peptides with higher Mascot scores. In summary, choice of LC conditions and fragmentation methods have direct bearing on numbers of peptides identified and their associated scores. These parameters can be leveraged for applications in quantitative proteomics and balanced with efficient use of instrument time.