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

• Parallel reaction monitoring was used with capillary zone electrophoresis.
• First use of CZE for bottom-up proteomic analysis of a human cell line.
• 468 proteins were identified in a single-shot analysis of the MCF-7 proteome.

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 h 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.