Robustness and Ruggedness of Isoelectric Focusing and Superficially Porous Liquid Chromatography with Fourier Transform Mass Spectrometry

John R. Corbett, Dana E. Robinson, Steven M. Patrie

Research output: Contribution to journalArticlepeer-review

Abstract

An investigation of a multidimensional proteomics workflow composed of off-gel isoelectric focusing (IEF) and superficially porous liquid chromatography (SPLC) with Fourier transform mass spectrometry (FTMS) was completed in order to assess various figures of merit associated with intact protein measurements. Triplicate analysis performed at both high and low FTMS resolutions on the E. coli proteome resulted in ∼900 redundant proteoforms from 3 to 95 kDa. Normalization of the chromatographic axis to identified proteoforms enabled reproducible physicochemical property measurements between proteome replicates with inter-replicate variances of ±3 ppm mass error for proteoforms <30 kDa, ±1.1 Da for proteins >30 kDa, ±12 s retention time error, and ±0.21 pI units. The results for E. coli and standard proteins revealed a correlation between pI precision and proteoform abundance with species detected in multiple IEF fractions exhibiting pI precisions less than the theoretical resolution of the off-gel system (±0.05 vs ±0.17, respectively). Evaluation of differentially modified proteoforms of standard proteins revealed that high sample loads (100s μgrams) change the IEF pH gradient profile, leading to sample broadening that facilitates resolution of charged post-translational modifications (e.g., phosphorylation, sialylation). Despite the impact of sample load on IEF resolution, results on standard proteins measured directly or after being spiked into E. coli demonstrated that the reproducibility of the workflow permitted recombination of the MS signal across IEF fractions in a manner supporting the evaluation of three label-free quantitation metrics for intact protein studies (proteoforms, proteoform ratios, and protein) over 102-103 sample amount with low femtomole detection limits.

Original languageEnglish (US)
JournalJournal of the American Society for Mass Spectrometry
DOIs
StateAccepted/In press - 2020

Keywords

  • chromatography
  • isoelectric focusing
  • label-free quantitation
  • mass spectrometry
  • proteoform
  • reversed-phase
  • top-down

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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