Development of Online pH Gradient-Eluted Strong Cation Exchange

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Development of an On-line pH Gradient-eluted Strong Cation Exchange NanoESI-MS/MS for Proteomic Analysis Facilitates Basic and Histidine-containing Peptides Identification Jingjing Xu, Jing Gao, Chengli Yu, Han He, Yiming Yang, Daniel Figeys, and Hu Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04000 • Publication Date (Web): 08 Dec 2015 Downloaded from http://pubs.acs.org on December 9, 2015

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Analytical Chemistry

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Development of an On-line pH Gradient-eluted Strong Cation Exchange NanoESI-MS/MS for Proteomic Analysis Facilitates Basic and Histidine-containing Peptides Identification Jingjing Xu1,2,3,#, Jing Gao1,2,5,#, Chengli Yu1,2,3, Han He1,2, Yiming Yang1, Daniel Figeys4,5,* and Hu Zhou1,2,5* 1

Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai, 201203, China 2

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai, 201203, China 3

University of Chinese Academy of Sciences, Chinese Academy of Sciences, China

4

Department of Biochemistry, Microbiology and Immunology, and Department of

Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada 5

SIMMUOMICS Laboratory, Joint Research Laboratory of Translational "OMICS"

between Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China and University of Ottawa, Canada

ACS Paragon Plus Environment

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ABSTRACT A novel one-dimensional on-line

pH

gradient-eluted strong cation exchange

(SCX)-nano-ESI-MS/MS method was developed for protein identification and tested with mixture of six standard proteins, total lysate of HuH7 and N2a cells, as well as membrane fraction of N2a cells. This method utilized an on-line nano-flow SCX column in a nano-LC system coupled with a nano-electrospray high-resolution mass spectrometer. Protein digests were separated on a nano-flow SCX column with a pH gradient and directly introduced into a mass spectrometer through nano-electrospray ionization. More than five thousand unique peptides were identified in each 90-min LC-MS/MS run using 500 nanogram protein digest either from total cell lysate or from membrane fraction. The unique peptide overlap between on-line strong cation exchange nano-ESI-MS/MS (SCXLC-MS/MS) and reverse phase nano-ESI-MS/MS (RPLC-MS/MS) is only ≤30%, which indicated these two methods were complementary to each other. The correlation coefficient of retention time and theoretical isoelectric point (pI) of identified peptides in SCXLC-MS/MS was higher than 0.4, which showed that peptides elution in SCXLC-MS/MS was dependent on their charge states. Furthermore, SCXLC-MS/MS showed identification capability for higher proportion of basic peptides compared to RPLC-MS/MS method, especially for histidine-containing peptides. Our SCXLC-MS/MS method is an excellent alternative method to the RPLC-MS/MS method for analysis of standard proteins, total cell and membrane proteomes.



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INTRODUCTION Mass spectrometry (MS) is a key analytical tool in proteomic research,1 and peptide identification is typically accomplished by tandem mass spectrometry (MS/MS).2 Proteomic samples are generally complex and their peptide constituents can cover a wide concentration range, and therefore, peptides separation in LC-MS/MS is a particularly analytical challenge. Reverse phase liquid chromatography (RPLC) is the most extensively used method for on-line peptides separation, because of its compatibility with MS.3-5 RPLC normally uses water and water miscible organic solvent (for example, acetonitrile, ACN) as mobile phases. Volatile acid (such as formic and acetic acid) is added into the mobile phase to render all of the component proteins and peptides positively charged and denatured and to reduce unwanted ionic interactions with the stationary phase.6 The increase in resolution provided by the RPLC separation enhances MS detection of sample components.6-8 Although the human proteome draft has been obtained with current mass spectrometry technology,9,10 innovative LC-MS/MS methods need to be developed to identify low-abundance proteins or proteins with extreme physichemical properties and to increase protein sequence coverage and identification capacity in proteomic analysis. Ion exchange chromatography is one of the most important analytical techniques for sample fractionation, and relies on electrostatic interactions between peptides and the surface charge of the ion exchange beads. Strong cation exchange (SCX) chromatography


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is the most commonly used fractionation method in proteomic research. For peptide fractionation, SCX chromatography is usually used in multidimensional chromotography and performed as the first dimension, not directly connected to the mass spectrometer. In these multidimensional LC-MS methods, salt gradient,11,12 pH gradient13,14 or combined pH and salt gradients15,16 are used for SCX chromatography. Salt-gradient elution is the most used method for SCX chromatography, while high salt concentration is not compatible with ESI-MS. Researchers have tried to improve the compatibility of SCX chromatography and ESI-MS. Huber and Buchmeister reported an on-line SCX-ESI-MS platform which can reduce adduct formation during the analysis of nucleic acids.17 Le Bihan et al reported an on-line SCXLC-MS/MS using a salt gradient from 0 to 1 M ammonium formate in 0.25 M FA, and demostrated that it was a useful method for proteomic studies.18 As far as we are aware, the report by Le Bihan et al. is the only manuscript reporting the direct coupling of SCXLC to mass spectrometer for proteomic analysis. An early study on 2D LC-MS using an on-line continuous pH gradient for SCX chromatography demonstrated that the continuous pH-gradient elution can minimize peptide overlap between adjacent fractions, increase sequence coverage and the concomitant confidence level in protein identification, and provide more basic peptides than salt-based elution.14 On the basis of those previous reports, we aimed to develop an on-line SCXLC-MS/MS method driven by pH gradient for proteomic analysis.



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Because of the hydrophobic nature of the SCX matrix (poly(styrene-divinylbenzene)), SCX chromatography behaves in a mixed-mode retention, including hydrophobic interactions and electrostatic interactions.19 Increasing the organic solvent proportion of the mobile phase can weaken the hydrophobic interactions between samples and the matrix.20-22 It had been demonstrated that peptide recovery from SCX increased up to five times by adding 25% ACN.21 Unfortunately, in multidimensional liquid chromatography (SCX-RPLC), the percentage of ACN added in the SCX elution buffers is usually less than 10%, because higher percentage of ACN is not compatible with peptide retention of the second dimension. Here we report a new method for on-line SCXLC-MS/MS based on pH gradient in 50% ACN. Using this approach, more than five thousand unique peptides were identified in SCXLC-MS/MS analysis. Comparison with RPLC-MS/MS indicates that only ≤30% of the peptides and ~55% of the proteins overlap between the two methods.

Moreover, SCXLC-MS/MS provides more identification of basic peptide

proportion, in particular histidine containing peptides than RPLC-MS/MS. Our SCXLC-MS/MS method is an excellent complement to RPLC-MS/MS method in proteomic analysis.

EXPERIMENTAL SECTION Chemicals and reagents


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Ammonium bicarbonate, Urea, Sodium carbonate, Malonic acid （M1296）, Formic acid (FA), Dithiothreitol (DTT) and Iodoacetamide (IAA) were obtained from Sigma (St. Louis, MO， USA). Water and Acetonitrile for LC-MS/MS were purchased from Avantor Performance Materials’ J.T. Baker (Center Valley, PA, USA). Trypsin was purchased from Promega (Madison, WI, USA). PLRP-S 1000 Å 10 µm beads and PL-SCX 1000 Å 10 µm beads were purchased from Agilent Technologies (Santa Clara, CA, USA). Standard proteins were obtained from Sigma: Fetuin from fetal calf serum (F3004), Lysozyme from chicken egg white (L7651),

Albumin from chicken egg white

(Ovalbumin, A7641), Carbonic anhydrase from bovine erythrocytes (C3934), Albumin from bovine serum (A9056), Serotransferrin from bovine (T1408). Standard protein in-solution digestion The 6 standard proteins were mixed in equal concentration, and dissolved in reducing buffer (8 M urea, 100 mM ammonium bicarbonate, pH 8.5). The standard proteins were digested using the reported procedures.23 N2a cell membrane sample preparation Mouse Neuro 2a (N2a) cells (American Type Culture Collection) were grown in DMEM including high glucose, 200 mM glutamine and 10% FBS (Invitrogen, Carlsbad, CA) and maintained in a humidified incubator at 37 °C and 5% CO2. A total of 2E8 N2a cells were washed three times with ice-cold PBS buffer and harvested in lysis buffer (2 M NaCl, 10 mM HEPES/NaOH, pH 7.4, 1 mM EDTA). The cells were sonicated mildly for 30 s at



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100 Watts (Scientz Biotech, Zhejiang, China) and centrifuged at 600 g for 5 min to remove intact cells. The suspension was centrifuged in a Himac CP 100MX preparative ultracentrifuge (Hitachi, Toyota, Japan) using the P40ST-2042 rotor at 200,000 g at 4°C for 20 min. The supernatant was discarded and the pellet was suspended in 12 mL of high pH buffer (100 mM Na2CO3, 1 mM EDTA, pH 11.3). After 30-min incubation on ice, the soluble material was removed by ultracentrifugation (as above). The pellet was suspended in 12 mL urea buffer (6 M urea, 100 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM EDTA), incubated on ice for 30 min, the soluble material was removed by ultracentrifugation. The pellet was washed with 10 mM Tris/HCl buffer (pH 7.6) twice and lysed with SDT buffer (4% SDS, 100 mM Tris/HCl pH 7.6, 100 mM DTT). Proteomic reactor and filter-aided sample preparation (FASP) digestion The HuH7 cell samples were processed by the centrifugal proteomic reactor24 with minor modifications. Briefly, 20 µg of protein from each sample was diluted and acidified by 1.2 mL of 1% formic acid, mixed with 10 µL of SCX slurry by vigorous vortexing. The samples were then centrifuged at 15 000 rpm, 4 min), and the resulting pellet (containing SCX beads and proteins) was washed 0.5% formic acid. The samples were reduced by mixing with 20 µL of 150 mM NH4HCO3, 50 mM DTT (shaking at 1000 rpm, 56°C, 15 min), and after reduction the DTT in the sample was diluted using 1.2 mL 0.5% formic acid and followed by centrifugation. The samples were then subjected to alkylation by mixing with 20 µL of 150 mM NH4HCO3, 100 mM iodoacetamide in darkness (15 min,


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room temperature), and the reaction was stopped by adding 1.2 mL of 0.5% formic acid containing 0.4 µg trypsin. After centrifugation, the resulting pellet was dissolved in 20 µL of 1 M NH4HCO3, and followed by trypsin digestion on a shaker at 1000 rpm at 37°C for 4 h. Finally, 300 µL of elution buffer (4 N NH3.H2O and 50% ACN) was added to elute the resulting peptides out of the SCX slurry. N2a total cell lysate and membrane lysate samples were digested by a filter-aided sample preparation (FASP) method according to a previously described method with some slight modifications.25 LC-MS/MS All

LC-MS/MS

experiments

were

performed

using

an

on-line

liquid

chromatography-tandem mass spectrometry (LC-MS/MS) setup which consisted of an Easy nano-LC system and a Q-Exactive mass spectrometer (Thermo, Bremen, Germany) equipped with a nano-electrospray ion source. The pump flow rate was set at 300 nL/min. The tryptic digested peptides were separated with a 90-min gradient. For each gradient either from strong cation exchange liquid chromatography or from reverse phase liquid chromatography, the peptide digests were analyzed by LC-ESI MS/MS at least three times to account for variability of protein identification results within the gradient. The mass spectrometry instrument parameters were set as followings: The temperature of the heated capillary was set at 320 °C and the source voltage was set at 2.4 kV. Orbitrap full scan automatic gain control target, 3e6; maximum injection time, 30 ms. MS2 scan



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automatic gain control target, 1e5; maximum injection time, 100 ms. The mass spectrometer fragmentation procedures were set up with the following parameters: one full MS scan was followed by 15 or 8 MS2 scans for the 15 or 8 most abundant precursor ions detected in the MS spectrum, the dynamic exclusion of 30 s to minimize repeated sequencing. The precursor ions were fragmented in the HCD cell by collision induced dissociation with normalized collision energy of 27%. The MS scan was acquired in the orbitrap mass analyzer with resolution 70,000 at m/z 200. The 445.120024 ion (Polysiloxane) was used as lock mass for real-time mass calibration. The SCXLC-MS/MS platform (Figure S1) consists of an Easy NanoLC system, a nanoflow SCX column (SCX, 75 µm×150 mm, 10 µm, 1000 Å, Agilent Technology) on-line with a nano-electrospray quadrupole-Orbitrap mass spectrometer (Q-Exactive). To make the SCX column, a 75-µm ID fused-silica microcapillary was pulled by a Model P-2000 laser puller (Sutter Instrument Co., Novato, CA), resulting in a tip with an opening ID of ~ 5 µm. The SCX column was packed in-house with a slurry of SCX beads all the way to the tapered tip using a high-pressure stainless steel vessel. As shown in Figure S1, the mobile phase A and B of the SCXLC-MS/MS platform were pH 2.5 and pH 8.5 in 50% acetonitrile (10 mM malonic acid, adjust by FA and NH3.H2O) respectively. The detailed steps to prepare mobile phase A for the SCX method was as followings: 1) add 5 mmol malonic acid (0.5203 g) into 250 mL water containing 0.1% formic acid; 2) after the malonic acid is dissolved completely, add 250 mL acetonitrile containing 0.1%


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fomic acid into the aqueous solution to make a final concentration of malonic acid at 10 mM in 50% acetonitrile; Mobile phase B for the SCX method is prepared as followings: transfer 250 mL buffer A to another bottle, and add NH3.H2O drop by drop until the pH reaches 8.5. The SCXLC gradient was: a 5-min gradient from 30% to 65% buffer B, a 72-min gradient from 65% to 100% buffer B, 13 min of 100% buffer B. Reverse phase chromatography was performed on a poly(styrene-divinylbenzene) copolymer (SDB) reverse-phase column (RP, 75 µm×150 mm, 10 µm, 1000 Å, Agilent Technology); mobile phase A was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. The RPLC gradient was as followings: a 1-min gradient from 2% to 5% buffer B, a 75-min gradient from 5% to 28% buffer B, a 1-min gradient from 28% to 90% buffer B, 4 min of 90% buffer B, a 1-min gradient from 90% to 0% buffer B and 8 min of 100% buffer A. Data analysis The MS data were analyzed using the software MaxQuant26,27 (http://maxquant.org/, version 1.3.0.5). Carbamidomethyl (C) was set as a fixed modification, and oxidation (M, +15.99492 Da) was set as a variable modification. Proteins were identified by searching MS and MS/MS data of peptides against a decoy version of 6 standard proteins or the International Protein Index (IPI) human/mouse database (version 3.87, European Bioinformatics Institute). Trypsin/P was selected as the digestive enzyme with two potential missed cleavages. The false discovery rate (FDR) for peptides and protein



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groups was rigorously controlled to be

Development of Online pH Gradient-Eluted Strong Cation Exchange

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