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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leukocyte, Cell Culture

DISEASE(S): Acute Leukemia

SUBMITTER: Zhuo Chen  

LAB HEAD: Arthur Salomon

PROVIDER: PXD002895 | Pride | 2022-02-28

REPOSITORIES: Pride