Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line

Abstract

We coupled capillary zone electrophoresis (CZE) with an ultrasensitive electrokinetically pumped nanospray ionization source for tandem mass spectrometry (MS/MS) analysis of complex proteomes. We first used the system for the parallel reaction monitoring (PRM) analysis of angiotensin II spiked in 0.45 mg/mL of bovine serum albumin (BSA) digest. A calibration curve was generated between the loading amount of angiotensin II and intensity of angiotensin II fragment ions. CZE-PRM generated a linear calibration curve across over 4.5 orders of magnitude dynamic range corresponding to angiotensin II loading amount from 2 amole to 150 fmole. The relative standard deviations (RSDs) of migration time were <4% and the RSDs of fragment ion intensity were ~20% or less except 150 fmole angiotensin II loading amount data (~36% RSD). We further applied the system for the first bottom up proteomic analysis of a human cell line using CZE-MS/MS. We generated 283 protein identifications from a 1 hour long, single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line digest, corresponding to ~80 ng loading amount. The MCF7 digest was fractionated using a C18 solid phase extraction column; single-shot analysis of a single fraction resulted in 468 protein identifications, which is by far the largest number of protein identifications reported for a mammalian proteomic sample using CZE.

Ion Activation Methods for Peptides and Proteins

“…[204][205] For example, over 10,000 peptides accounting for 2,000 proteins were identified from a 100 minute analysis of a HeLa cell digest using capillary zone electrophoresis with CID as the ion activation method. 204 Along those same lines, CZE-MS/MS (based on CID) was used to identify over 2,000 phosphopeptides from an MCF-10A cell line. 205 These are just a few examples demonstrating the utilization of CID data for high throughput identification of peptides.…”

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

“…10 However, the performance of CZE–MS/MS for shotgun proteomics has been disappointing. To date, single‐shot CZE–MS/MS proteomic analyses have enabled the identification approximately 1000 peptides and 300 proteins,10, 11 which is at least ten times less than the state‐of‐art RPLC–MS/MS system 12–15…”

Over 10 000 Peptide Identifications from the HeLa Proteome by Using Single‐Shot Capillary Zone Electrophoresis Combined with Tandem Mass Spectrometry

“…[9,10] However, the performance of CZE-MS/MS for shotgun proteomics has been disappointing. To date, singleshot CZE-MS/MS proteomic analyses have enabled the identification approximately 1000 peptides and 300 proteins, [10,11] which is at least ten times less than the state-ofart RPLC-MS/MS system. [12][13][14][15] Two obstacles limit CZE-MS performance.…”