Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry.
The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ∼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in <15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.
UT Southwestern Medical Center, Dallas, Texas 75390-9185, United States.
Michael J
MJ
Roth
Daniel A
DA
Plymire
Audrey N
AN
Chang
Jaekuk
J
Kim
Erica M
EM
Maresh
Shane E
SE
Larson
Steven M
SM
Patrie
D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006367:HeLa Cells; D006657:Histones / Q000032:analysis; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D025801:Ubiquitin / Q000032:analysis
D006657:Histones; D011506:Proteins; D025801:Ubiquitin