This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In shotgun proteomics, reverse phase nanoflow HPLC is widely used as a method of separating peptides prior to analysis by mass spectrometry. The chromatographic separation of peptides enables the analysis of complex peptide samples that would otherwise not be possible. We are interested in the effect of altering nanoLC parameters, such as column length and gradient length, on peak capacity and peptide identifications. We are also interested in whether obtaining optimal chromatographic peak capacity results in optimal peptide identifications.