Standard Pressure LC-MS/MS Using 1.9um C18 Particles Column at Room Temperature

Although the benefits of reduction of the size of reversed phase particles are established to provide increased sequencing depth and improved chromatography in LCMS experiments, the wide-scale adoption of optimally sized small particles in reversed-phase columns has been hampered by the necessity for specialized equipment such as ultra-high pressure liquid chromatography or a customized column heating apparatus. Here, we introduce a new strategy to routinely fabricate a 50 cm-long, 1.9 µm particle C18 column and extensively characterize the performance of this column. This column was packed under 100 Bar and routinely utilized on a standard quarternary HPLC at pressures below 300 Bar. Expanding the depth of sequencing of peptides that show a statistically significant quantitative change arising from a biological stimulation is critical. Compared with traditional C18 columns packed with 3 µm particles, the column with the 1.9 µm particles operated with a standard HPLC could detect 330% more peptides with statistically significant changes from differentially stimulated T cells. This improved column fabrication methodology provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize sequencing depth, dynamic range, sensitivity, and reproducibility. This study also highlights the importance of the statistical analysis of quantitative proteomic data instead of a sole focus on peptide spectrum match yields.