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

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.

Online μSEC2-nRPLC-MS for Improved Sensitivity of Intact Protein Detection of IEF-Separated Nonhuman Primate Cerebrospinal Fluid Proteins

“…Past proteomics investigations on CSF (e.g., 2D electrophoresis) have shown that removal of abundant serum proteins, many of which are mid-to-high MW (i.e., >30 kDa) (e.g., albumin (66 kDa), α1-acid glycoprotein (44 kDa), α1-antitrypsin (45 kDa), transthyretin (55 kDa), transferrin (80 kDa), haptoglobin (86 kDa), IgGs (150 kDa), and α2-macroglobulin (800 kDa)), ,,, results in substantial improvement in the detection of CSF proteins that are lower in concentration and MW. , Furthermore, <30 kDa is a common working range for large label-free quantitation investigations by intact protein MS. − Therefore, we next sought to determine if the μSEC 2 -nRPLC-MS platform would impart similar benefits for CSF-IEF fractions that commonly present with these HMW proteins that can overwhelm the IEF buffering capacity at high concentrations resulting in their poor fractionation . This was accomplished by integration of the 1000 Å pore size = 200 mm LP-μSEC column downstream of HP-μSEC and the first RP-trap yet upstream of the second RP-trap and nRPLC-MS (Figure ).…”

“…16,17 Furthermore, <30 kDa is a common working range for large label-free quantitation investigations by intact protein MS. 53−56 Therefore, we next sought to determine if the μSEC 2 -nRPLC-MS platform would impart similar benefits for CSF-IEF fractions that commonly present with these HMW proteins that can overwhelm the IEF buffering capacity at high concentrations resulting in their poor fractionation. 58 This was accomplished by integration of the 1000 Å pore size = 200 mm LP-μSEC column downstream of HP-μSEC and the first RP-trap yet upstream of the second RPtrap and nRPLC-MS (Figure 1). The platform's modularity, specifically the ease of SEC-MS implementation, enables rapid evaluation of columns, flow conditions, and switching valve timing for loading of different SEC fractions onto traps.…”

Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins

“…14, 15 Furthermore, < 30 kDa is a common working range for large label-free quantitation investigations by intact protein MS. 36–39 Therefore, we next sought to determine if the SEC 2 -nRPLC-MS platform would impart similar benefits for CSF-IEF fractions that commonly present with these HMW proteins that can overwhelm the IEF buffering capacity at high concentration resulting in their poor fractionation ( Supplementary Figure 6-left ). 52 This was accomplished by integration of the 1000 Å pore size, ℒ = 200 mm LP-μSEC column downstream of HP-SEC and the first RP-trap yet upstream of the second RP-trap and nRPLC-MS ( Figure 1 ). The platform’s modularity, specifically ease of SEC-MS implementation, enables rapid evaluation of columns, flow conditions, and switching valve timing for loading of different SEC fractions onto traps.…”

High-throughput proteomics: a methodological mini-review

“…Although 2D-PAGE has traditionally been used as a standard procedure for proteomics research, gel-based techniques tend to be labor-intensive and time-consuming, and are therefore not suitable for high-throughput proteomics. By contrast, LC or high-performance liquid chromatography (HPLC) allows continuous separation of thousands of proteins from complex mixtures and can be combined with MS as LC-MS for increased throughput 19 – 21 . Among them, Reversed-phase liquid chromatography (RPLC) is the most commonly used LC-based separation platform.…”