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. 2014 Aug 5;86(15):7899-906.
doi: 10.1021/ac501836k. Epub 2014 Jul 9.

Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-Down Proteomics

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Free PMC article

Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-Down Proteomics

Lichen Xiu et al. Anal Chem. .
Free PMC article

Abstract

One of the challenges in proteomics is the proteome's complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.

Figures

Scheme 1
Scheme 1. Comparison of Chromatographic Methods for Separations Based on Differences in Polarity
Green and red arrowheads indicate the direction of gradient polarity during elution. HIC, hydrophobic interaction chromatography; RPC, reverse phase chromatography; HILIC, hydrophilic interaction chromatography; NPC, normal phase chromatography.
Figure 1
Figure 1
Overlay of HIC chromatograms of individual standard proteins in different mobile phases containing (a) ammonium sulfate (AS) and (b) ammonium tartrate (AT). Conditions: PolyPROPYL A column, 100 mm × 4.6 mm i.d., 3 μm, 1500 Å; MPA, 1.8 M salt aqueous solution at pH 7.0; MPB, 20 mM salt aqueous solution at pH 7.0; column temperature, 25 °C; UV detection, 280 nm; flow rate, 1 mL/min; gradient, 30 min from 100% MPA to 100% MPB. The gray dash line refers to the MPB percent in the gradient profile. Apr, aprotinin; Cyt, cytochrome C; Myo, myoglobin; Oval, ovalbumin; BSA, bovine serum albumin; RiB, ribonuclease B; RiA, ribonuclease A; Chy, α-chymotrypsin; ChA, α-chymotrypsinogen A; Con, conalbumin.
Figure 2
Figure 2
HIC separations of standard protein mixtures with ammonium sulfate (a,b), ammonium tartrate (c,d), and ammonium acetate (e,f). HIC UV-chromatograms of 4-mix sample is shown in the left column (a, c, and e) and 10-mix sample is depicted in the right column (b, d, and f). Conditions: same as shown in Figure 1.
Figure 3
Figure 3
Evaluation of MS-compatibility of HIC salts. Representative mass spectra of ribonuclease A in HIC buffers: (a) 1.8 M AS, (b) 1.8 M AT, desalted by ultracentrifugal device and RPC. NL, normalized level.
Figure 4
Figure 4
Separation of standard protein mixtures by HIC using ammonium tartrate as salt (a and b) and RPC (c and d), suggesting the orthogonality between HIC and RPC. (a) and (c), 4-mix (Myo, Oval, BSA, ChA). (b and d) 6-mix (Cyt, RiB, Myo, RiA, Oval, Con). HIC conditions, same as shown in Figure 1. RPC conditions, PicoFrit column (New Objective) PLRP-S, 100 μm i.d. × 100 mm, 5 μm, 1000 Å; MPA, water with 0.25% formic acid; MPB, acetonitrile with 0.25% formic acid; column temperature, 25 °C; flow rate, 500 nL/min. Gradient: Initial 15 min isocratic segment of 5% MPB, followed by linearly increasing MPB to 25% in 10 min, then to 60% linearly in 45 min, to 95% in the next 5 min, and finally decreased to 5% in 5 min. Total: 80 min.
Figure 5
Figure 5
HIC-RPC separation and MS analysis of E. coli cell lysate samples. Representative RPC and MS data from one HIC fraction are shown. (a) UV-chromatogram obtained for E. coli cell lysate by HIC separation with ammonium tartrate as the gradient salt HIC conditions, same as shown in Figure 1, except the gradient profile simplified to a 30 min linear gradient from 100% MPA to 100% MPB. (b) RPC-MS TIC for HIC fraction 2 after ultracentrifugal desalting. RPC conditions, same as shown in Figure 3. (c and d) Representative mass spectra for three E. coli proteins observed in the HIC-RPC MS platform with charge state distributions (no tartrate adduction) and unit mass isotopic resolution on a chromatography time scale on Q Exactive.
Figure 6
Figure 6
Online RPC/MS/MS protein identifications by HCD for HIC fraction 2. Representative MS/MS spectra and sequence maps of identified proteins with b/y ion cleavages and P values for protein identification. The insets highlight the isotopic resolution for representative fragments at m/z 916 and 2024, respectively.

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