Efficient Exploitation of Separation Space in Two-Dimensional Liquid

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Efficient exploitation of separation space in two-dimensional liquid chromatography system for comprehensive and efficient proteomic analyses Hangyeore Lee, Dong-Gi Mun, Jeong Eun So, Jingi Bae, Hokeun Kim, Christophe Masselon, and Sang-Won Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03366 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Analytical Chemistry

Efficient exploitation of separation space in two-dimensional liquid chromatography system for comprehensive and efficient proteomic analyses Hangyeore Lee†, Dong-Gi Mun†, Jeong Eun So†, Jingi Bae†, Hokeun Kim†, Christophe Masselon‡, §, ∥ , and Sang-Won Lee*, † †

Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea

‡

Université Grenoble Alpes, F-38402 Saint-Martin-d'Heres, France

§

CEA, Institut de Biosciences et de Biotechnologie de Grenoble, Biologie à Grande Echelle, F-38054 Grenoble, France

∥

INSERM, U1038, F-38054 Grenoble, France

ABSTRACT: Proteomics aims to achieve complete profiling of the protein content and protein modifications in cells, tissues and biofluids, and to quantitatively determine changes in their abundances. This information serves to elucidate cellular processes and signaling pathways, and to identify candidate protein biomarkers and/or therapeutic targets. Analyses must therefore be both comprehensive and efficient. Here, we present a novel online two dimensional reverse phase/reverse phase liquid chromatography separation platform, based on a newly developed online non-contiguous fractionating and concatenating device (NCFC fractionator). In bottom-up proteomics analyses of a complex proteome, this system provided significantly improved exploitation of the separation space of the two RPs, considerably increasing the numbers of peptides identified compared to a contiguous 2D-RP/RPLC method. The fully automated online 2D-NCFC-RP/RPLC system bypassed a number of labor-intensive manual processes required with the previously described offline 2D-NCFC RP/RPLC method, and it thus offers minimal sample loss in a context of highly reproducible 2D-RP/RPLC experiments.

Many liquid chromatography techniques have been developed over the years to extend proteome coverage in the drive towards ever more comprehensive characterization of the proteomes of biological systems, while also increasing sample throughput by exploiting liquid chromatography tandem mass spectrometry (LC-MS/MS) technological platforms1. However, a number of challenges remain, and no-one has yet claimed to provide complete characterization of any proteome due to the high complexity of biological samples, potentially containing tens of thousands proteins in detectable amounts, possibly harboring various modifications, and covering a dynamic range of greater than ten orders of magnitude2,3. Two-dimensional liquid chromatography (2DLC) techniques exploit two different separation dimensions (e.g. ion exchange and reverse phase) in a single setup. Many of these setups have been tested in LC-MS/MS experiments, applying a first dimension separation as a sample fractionation method to reduce sample complexity prior to separation in the second dimension (typically using low-pH reverse–phase chromatography) in preparation for tandem mass spectrometry (MS/MS) 4-6 . A highly efficient two-dimensional LC system will combine high orthogonality (i.e., dissimilarity or distinct selectivity) between the two separation methods, and high chromatographic peak capacity (i.e., high chromatographic resolution) in both separation dimensions. A commonly used online

2DLC configuration combines strong cation exchange (SCX) in the 1st dimension and RP chromatography in the 2nd dimension. The good results obtained with this system are largely due to the high orthogonality between the two separation modes7. More recently, both online and offline 2D-RP/RPLC methods employing two RPs at different pHs (e.g. high- and low-pH) have been described8-11. Instead of using different stationary phases to alter selectivity, different pH conditions can be used for the two RP separations in 2D-RP/RPLC. The pH of the buffer modifies the solution phase charge states (and thereby hydrophobicities) of peptides present based on their isoelectric points. The selectivity of the two RP separations is similar but non-identical, but this is (at least partially) counterbalanced by the high chromatographic resolutions possible in both dimensions. Indeed, 2D-RP/RPLC was demonstrated to enhance peptide coverage in LC-MS/MS experiments and to provide the highest practical peak capacity when a number of 2DLC techniques were compared12,13. In a conventional implementation of 2D-RP/RPLC, eluates from the 1st RPLC (e.g. high-pH or mid-pH) are collected contiguously for separation by the 2nd RPLC (e.g. low-pH). This is problematic as, since the two RPs are related, their relative separation power is only semi-orthogonal (i.e., elution orders for peptides in the two RPs will not be completely dissimilar)13,14.

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Figure 1. Comparison of offline and online NCFC methods. A detailed workflow illustrating how online NCFC was performed can be found in Figure S1. Non-contiguous fractionation and concatenation of initial 50 fractions to obtain 10 NCFC fractions was illustrated.

This reduces the extent to which the separation spaces of the two systems are used. To overcome this limitation, offline 2DRP/RPLC methods based on a non-contiguous fractionating and concatenating (NCFC) strategy which helped to increase the effective orthogonality between the high-pH RPLC and the low-pH RPLC by shuffling the peptides as they eluted from the high-pH RPLC were introduced15-17. Figure 1A illustrates a typical offline 2D NCFC-RP/RPLC approach. The sample was initially fractionated into a large number of fractions (e.g. 50 fractions) using the high-pH RPLC coupled to a fraction collector and the initial fractions were defined into five groups of fractions (early, mid-early, middle, mid-late, and late elution), followed by pooling five non-contiguous fractions (i.e. one from each of the five groups) to produce 10 concatenated fractions. Each of the pooled fractions was then individually subjected to offline desalting, drying, and reconstitution in injection buffer before autosampler injection for low-pH RPLC separation. The results demonstrated quasi-optimal utilization of the separation space in the second RP dimension, thus producing efficient and orthogonal sample fractionation. The main drawback with this process is that it generally requires a relatively large amount of sample for the high-pH NCFC fractionation as preparative columns are used to allow for the inevitable sample loss associated with the various offline steps18. In this study, we developed an NCFC device (Figure 1B) that performs the NCFC in the mid-pH RPLC separation and combined the system online with a dual-online low-pH RPLC, a technique which has previously been used in high-throughput LC-MS/MS experiments19. The combined system, 2D-NCFCRP/RPLC, efficiently performed online 2D-RP/RPLC in NCFC mode with full automation, thus bypassing the labor-

intensive manual steps required with offline 2D-NCFCRP/RPLC. The results demonstrated that peptide fractions of uniform complexity were produced by the 1st dimension RPLC system, and that the separation space of the 2nd dimension RPLC setup was fully exploited. Thanks to these two characteristics, more comprehensive peptide identifications were possible than with a contiguous online 2D-RP/RPLC method.

EXPERIMENTAL SECTION Chemicals and materials. LC-MS-grade water and acetonitrile (ACN) were purchased from Fisher Scientific (Fair Lawn, NJ). Formic acid, trifluoroacetic acid (TFA), triethylammonium bicarbonate (TEAB) buffer and ammonium bicarbonate (ABC, NH4HCO3) were purchased from SigmaAldrich (St Louis, MO). Sequencing-grade modified porcine trypsin was purchased from Thermo Scientific (Rockford, IL). A fresh frozen tumor tissue was obtained from a patient with diffuse-type gastric cancers (M/38) at the time of performing curative surgery at the Asan Medical Centre in Korea. The patient gave written informed consent for use of these tissues in this study. The study protocol was approved by the local ethics committee (NCCNCS-120581). Sample preparation. Peptides were prepared from the tissue sample as described in Lee et al.20. Briefly, the tissue was washed in PBS buffer on ice to remove blood and then cryopulverized into a fine powder using a Cryoprep pulverizer (CP02 Cryoprep, Covaris, Woburn, MA) according to the manufacturer’s instructions. The pulverized tissue was then suspended in 330 µL of lysis buffer (4% SDS in 0.1M TrisHCl,


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Figure 2. Schematic representation of the online 2D-NCFC-RP/RPLC system. The lines on the five 2-position valves labeled S, F, L, CS and DC denote position 1 (solid lines) and position 2 (dotted lines). Details of the operations performed during sample fractionation are given in Methods.

pH 7.6, phosphatase inhibitor) and lysed with a focusedultrasonicator (S220, Covaris, Woburn, MA). A second round of lysis was performed with a probe sonicator (Q55 sonicator, Qsonica, Newtown, CT) to achieve complete lysis. The tissue lysate was reduced at 37 °C for 45 min after adding DTT (0.1 M final concentration), then a slightly modified filter-aided sample preparation digestion protocol was applied21. The modified protocol was as follows: the reduced proteins were mixed with 200 µL of 8 M urea solution (8 M urea in 0.1 M Tris-HCl, pH 8.5) on a membrane filter unit (Microcon device, YM-30, Millipore, MA) and then centrifuged to remove SDS. Iodoacetamide (IAA) solution (100 µL of 500 mM) was added to alkylate cysteine residues during incubation for 20 min at room temperature. The sample was washed once again with 200 µL of 8 M urea, and then with 100 µL of 50 mM ABC. Trypsin was added to the sample on the filter unit at a trypsinto-protein ratio of 1:50 (w/w). Digestion was allowed to proceed at 37 °C for 12 hours. Additional trypsin was then added to the sample at a trypsin-to-protein ratio of 1:100. Digestion was pursued at 37 °C for a further 6 hours. The peptide concentration of the resultant sample was estimated using a BCA assay (BCA Protein Assay Kit, Pierce, Rockford, IL). The sample was then vacuum dried (Eppendorf concentrator plus, Eppendorf, Hamburg, Germany) and stored at – 80 °C until LC-MS/MS experiments were performed. Online 2D-NCFC-RP/RPLC system. The online 2DNCFC-RP/RPLC system is schematically illustrated in Figure 2. The system consisted of two nanoflow binary pumps (Binary pump 1 and Binary pump 2; Ultimate 3000 NCP-3200RS, Dionex, Germering, Germany), two isocratic pumps (Isocratic pump 1; Model 65 DM, ISCO, Lincoln, NE. And Isocratic pump 2; Agilent1100, G1312A, Palo Alto, CA), an autosampler (Ultimate 3000 WPS-3000TPL RS, Dionex, Germering, Germany), and a valve module. Mid-pH solvents, solvents A and B, were 10 mM TEAB in water (pH 7.5) and 10 mM TEAB in 99% ACN (pH 7.5), respectively. Low-pH

solvents, solvents C and D, were 0.1% formic acid in water and 0.1% formic acid in ACN, respectively. Binary pump 1 was used to inject samples (onto either the mid-pH RP column or SPEs) and to generate the gradient for mid-pH RPLC. TEAB solvents (pH 7.5) were used instead of the conventional ammonium formate solvents (pH 10) as they had a less detrimental effect on columns while retaining similar orthogonality between the two RPLCs19. Binary pump 2 was used to generate the gradient for low-pH RPLC. Isocratic pump 1 was used to equilibrate both SPEs and RP COLs, while Isocratic pump 2 was used to dilute the NCFC fractions to reduce organic content to under 3%, while simultaneously acidifying the sample through the addition of TFA. This ensured effective loading and desalting in SPE columns. The valve module was composed of six ultra-high pressure, nanovolume valves (VICI, Houston, TX): a function selection valve (F, 6-port/3-channel/2-position, C72MX-4676), two NCFC valves (NCFC1 and NCFC2, 11-port/1-channel/multiposition, C85H-6670SH), a connection valve (C, 4-port/2channel/2-position, C72MX-4674), a column selection valve (CS, 4-port/2-channel/2-position, C72MX-4674), and a dual column valve (DC, 10-port/ 5-channel/ 2-position, C72MX4670, VICI). Each of the six valves was equipped with a universal actuator (EUHC, VICI, Houston, TX) for automated timed control of its valve position. The solid and dotted lines in Figure 2 indicate positions 1 and 2 for the five switching valves. The F-valve, on which capillary RP column for midpH RPLC (mRP COL, 50 cm × 150 µm) was installed, was used to select the separation mode: 1-dimensional RPLC (F(2) position, bypassing the mid-pH RP COL), 2-dimensional RPLC (F(1) position), or NCFC fraction injection (F(2) position). Two SPE columns (SPE1 and SPE2, 3 cm × 150 µm) and two capillary analytical RP columns (COL1 and COL2, 150 cm × 75 µm ) were installed on the DC-valve so as to allow independent and parallel utilization of the two SPE/COL pairs (SPE1/COL1 and SPE2/COL2) by using a single switching valve, as previously described20. DC(1) is the position for



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sample injection and online desalting, while DC(2) is the position for RPLC separation. The two series of 10 ports on the NCFC valves (Figure 1 and 2) were connected by ten fraction loops (inner volume ca. 6 µL, 19.2 cm × 200 µm). The elution port of the F-valve was connected to the central port on valve NCFC1 by a fused silica capillary (15 cm × 50 µm), while the central port on valve NCFC2 was connected to a port on the Cvalve by a fused silica capillary (15 cm × 50 µm), as shown in Figure 2. A tee connector was used to combine flows from the outlet of the NCFC fractionator and Isocratic pump 2 and to direct the combined flow to the C-valve. The C-valve either directs the fractions to the CS-valve (when in position C(2)) or sends solvents to the waste line (in position C(1)) during NCFC fractionation. The CS-valve in turn either directs the NCFC fractions to SPE1 (when in position CS(1)) or to SPE2 (when in position CS(2)) when the DC-valve is in position DC(1). When the DC-valve is in position DC(2), the CS-valve also directs the gradient produced by Binary pump 2 to either SPE1/COL1 (when in position CS(2)) or SPE2/COL2 (when in position CS(1)). The positions of the six valves making up the module were automatically time-controlled by asserting (e.g. grounding) the digital I/O pins on the universal actuators using the electronic switches of an external switching device (UCI-50, Dionex, Germering, Germany) controlled by Chromeleon software (Dionex, Germering, Germany). A signal relay (DS1E-M-DC5 V, Panasonic Electric Works Co, Osaka, Japan) was inserted between each of the universal actuator’s digital I/O pins and its corresponding electronic switch. The columns (mid-pH RP COL, RP COL1/2, and SPE1/2) were manufactured in-house with C18 particles (Jupiter, 3 µm, 300 Å, Phenomenex, Torrance, CA) using a previously described slurry packing method22. The temperature of the midpH RP COL and the two COLs was set to 60 °C using semiflexible column heaters, as described previously23. Online CF and NCFC methods. For both online fractionation procedures (CF and NCFC), a 20-µg peptide sample was injected into the mid-pH RP COL using an autosampler (Ultimate 3000 WPS-3000TPL RS, Dionex, Germering, Germany). During sample injection, Binary pump 1 supplied 1% solvent B at a flow rate of 1.0 µL/min for 8 min while the valves were in S(1), F(1), NCFC1(1), NCFC2(1), C(1) positions. During the online fractionation, the CS- and DC- valves remained in CS(1) and DC(2) positions, respectively, to equilibrate both SPEs and COLs with solvent C. After sample injection, a 100min gradient (1-50% solvent B at a flow rate of 500 nL/min) from Binary pump 1 was used to perform the 1st dimension mid-pH RP separation. For online CF fractionation, a total of ten 10-min eluates were sequentially collected in the ten fraction loops by steprotating the two NCFC valves synchronously every 10 minutes. For online NCFC fractionation, a total of fifty 2-min eluates were sequentially collected into the 10 fraction loops by step-rotating the two NCFC valves synchronously every 2 minutes. Thus, after 20 minutes, the eleventh 2-min eluate was collected in the first fraction loop, where it was combined with the first 2-min eluate (Figure S1). Thus, at the end of the 100min gradient, each of the 10 fraction loops contained five noncontiguous fractionated and concatenated eluates (eluates 111-21-31-41, eluates 2-12-22-32-42, etc.), which correspond to the 10 NCFC fractions. Dual-online RP/RPLC experiments on fraction samples. Each of the 10 fractions (either CF or NCFC fractions) was

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then injected onto an SPE column with the valves in S(2), F(2), C(2) and DC(1) positions. The position of the CS-valve was switched to direct the fraction to either SPE1 or SPE2. The positions of the two NCFC valves were synchronously switched to select the fraction to be injected. To reduce acetonitrile content and acidify each fraction, to make it compatible with the 2nd dimension low-pH separation, a dilution solvent (0.2% TFA in water) was supplied through Isocratic pump 2 at a flow rate of 2.7 µL/min. In the tee, this solvent was mixed with the fraction (in solvent A and B) arriving at a flow rate of 300 nL/min from Binary pump 1, thus producing a 10× dilution. After injection of a fraction into an SPE column, the DCvalve was switched to position DC(1) and the RP gradient (4%–5% solvent D over 10 min, 5-45% over 330 min, 45%– 80% over 5 min, 80% for 20 min and 1% for 5 min) from Binary pump 2 was directed into the appropriate SPE/COL at a flow rate of 300 nL/min to perform the 2nd RPLC separation. Detailed information on valve positions and other experimental sequences and settings used in the online 2D-NCFCRP/RPLC experiments are summarized in table S1. MS analysis. Peptide masses were measured using a QExactive orbitrap mass spectrometer (Thermo Electron, San Jose, CA), which was coupled to the online 2D-NCFCRP/RPLC system by a specially designed two-position valve, as previously described24. The spray voltage was kept at 2.4 kV and the temperature of the desolvation capillary was maintained at 250 °C. Full MS scans were acquired with an m/z range of 400-2,000 Th with a resolution of 70,000. The target values for automatic gain control (AGC) and maximum ion injection time (IT) were 1.0 × 106 and 20 ms, respectively. For data-dependent MS/MS experiments, up to ten of the most abundant ions were selected for fragmentation with an isolation window of 0.8 Th and an exclusion duration of 30 s. Higher energy collisional dissociation was used for fragmentation with a normalized collision energy of 25. Ions with a charge state of 1 were excluded and the intensity threshold to initiate fragmentation was set at 1.7 × 104. MS/MS scans were acquired at a resolution of 17,500, target AGC value was set to 1.0 × 106, and a maximum IT of 60 ms was defined. The LCMS/MS data have been deposited to the ProteomeXchange Consortium via the PRIDE25) partner repository with the dataset identifier PXD004710 (ID: [email protected], PWD: UtBWWnUN). Analysis of LC-MS/MS data. Tandem mass spectrometry (MS/MS) data were processed using PE-MMR (http://omics.pnl.gov/software/PEMMR.php) to correct and refine precursor masses26. MSGF+ (v9387, http://omics.pnl.gov/software/ms-gf)27 was used to search the resultant MS/MS data (as mgf files) against a composite database containing the Uniprot reference human database (May 2013 release; 90,219 entries) and a list of common contaminants (179 entries). The precursor mass tolerance was set to 10 ppm and the number of tryptic termini was 1. Carbamidomethylation to cysteine was set as a fixed modification, and methionine oxidation was set as a variable modification. Peptide spectrum matches at a FDR of 0.01 were retained for further analysis28. Briefly, the non-redundant peptides identified were used to infer protein groups by a bipartite graph analysis29 using an in-house software30. Protein groups of two or more non-redundant peptides were reported by the representative protein of protein group being selected as the protein with the


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highest number of peptides. Normalized elution time (NET) utility was used to calculate the experimental NETs of the identified peptides31.

RESULTS AND DISCUSSION Development of an online 2D-NCFC-RP/RPLC system directly coupled to a mass spectrometer. To achieve online NCFC of eluates from the first RP separation dimension, we developed an NCFC fractionator based on two interconnected 10-position selector valves. As shown in Figure 1B (and Figure S1), eluates from the mid-pH RP column were sequentially deposited in each of the 10 fraction loops in turn for a defined period of time (e.g. 2 minutes) by synchronously step-rotating the two selector valves. After the first collection cycle - once all ten fraction loops contained an eluate - the NCFC fractionator continued synchronously rotating the two valves stepwise to non-contiguously concatenate the fractions eluted from the mid-pH RP column. Thus, the 11th eluate was concatenated with the 1st eluate, the 12th with the 2nd, and so on. After five full cycles, each of the 10 fraction loops contained 5 noncontiguous eluates (or pre-fractions) from the mid-pH RPLC separation. By using different sequences of synchronous step rotation, different numbers of NCFC fractions or different concatenation schemes can be generated. Thus, a two-step rotation (e.g., stepping position 1→3→5⋅⋅⋅→9→1 instead of position 1→2 →3⋅⋅⋅→10→1) would produce 5 NCFC fractions. Through these adaptations, various fully automated NCFC fractionations can be achieved, including random sequence concatenation. The system is therefore compatible with a variety of samples and applications. The NCFC fractionator can also be operated in contiguous fractionation (CF) mode by collecting the eluates from the mid-pH RP column sequentially in the 10 fraction loops in a single cycle, as described in the Methods section. Figure 2 shows a schematic diagram of the fully automated 2D-NCFC-RP/RPLC system in which the NCFC fractionator was intercalated within a dual-online reverse phase liquid chromatography system (DO-RPLC)20. The DO-RPLC system combined two sets of analytical (RP COL1 and RP COL2) and solid phase extraction (SPE1 and SPE2) columns which operate in parallel or alternately. This system was previously demonstrated to increase the experimental duty-cycle 2-fold while also allowing back-flush sample injection for fast and narrow-zone sample injection after online desalting. Offline fractionation methods using conventional autosampler injection have been linked to significant sample loss, reducing protein identification by up to 44.5% compared to online fractionation methods included in multidimensional protein identification technology (MudPIT)18. By coupling the NCFC fractionator online to the DO-RPLC, the 10 NCFC fractions could be automatically produced and sequentially analyzed by the two sets of analytical and solid phase extraction (SPE) columns after an online desalting step, thus bypassing all the manual steps (fraction concatenation, desalting, drying, reconstitution and autosampler injection) involved in offline 2D-RP/RP. Comparison of CF mode with NCFC mode for online 2D-RP/RPLC. The new online 2D-RP/RPLC system can be operated in either CF or NCFC modes by choosing to collect eluates either by contiguous fractionation, cycling only once

through the 10 fraction loops or in non-contiguous fractionation by concatenating eluates through multiple cycles. The number of peptides identified when the online 2D-RP/RPLC system was operated in CF mode was reported in a heat map (Figure 3A), where peptide identifications tend to concentrate along the diagonal line. This heat map clearly shows the semiorthogonal relationship between mid-pH and low-pH RPLC, which results

Figure 3. Comparison of online 2D-RP/RPLC operated in CF mode or NCFC mode. Heat maps indicate the number of peptides identified after online 2D-RP/RP using the CF (A) and NCFC (B) methods. The bold lines delimit the separation space where all fractions produced more than 50 peptide identifications per NET bin (0.01). The y-axis shows the NET for peptides on the low-pH RP for each mid-pH RP fraction (x-axis). (C) Comparison of number of peptides identified in each fraction by the CF and NCFC methods. (D) Venn diagram showing the number of nonredundant peptides identified by the CF and NCFC methods.

in limited use of the available separation space (Figure S2). The use of high-pH RPLC (i.e. by using pH 10 buffer of ammonium formate) for the 1st dimension in CF mode improved the orthogonality to some extent, but still suffered the limited use of separation space of the 2nd dimensional low-pH RPLC (Figure S3). When the same 2D-RP/RPLC system (i.e. mid-pH/low-pH RPLC) was operated in NCFC mode with five fractionation cycles, the resultant heat map (Figure 3B and Figure S4) shows a significant increase in the use of the separation space, with almost full coverage of the normalized elution time (NET) range for peptides between 0.1 and 0.7 across all 10 fractions32. The proportion of separation space for which more than 50 peptides were identified per NET bin (0.01) was significantly larger when the 2D-RP/RPLC system was operated in NCFC mode. Compared with the results from operation in the CF mode, the NCFC mode produced more uniform numbers of peptides identified across the 10 fractions (Figure 3C). The orders in which peptides eluted in the 1st RP dimension mirrors their intrinsic hydrophobicity in mid-pH conditions, most commonly resulting in only a small number of peptides eluting at either end of the LC gradient (fractions 1, 2, 9, and 10 in Figure 3A). It is therefore very difficult (or even impossible) to produce uniform peptide fractionation by contiguous collection of RPLC eluates. By concatenating the peptides as they elute in a non-contiguous manner, each fraction will contain peptides eluting early, middle or late, thus producing more



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uniform peptide complexities. This uniformity is clearly shown by the similar number of peptides identified in all ten fractions, and the uniformity of the pattern on the heat maps showing the number of peptides identified. Despite using the same amount of sample (20 µg) and the same analytical throughput on the same RP columns, the NCFC mode identified twice as many peptides as the conventional CF mode (i.e., 126,814 vs. 59,678 non-redundant peptides at 1% false discovery rate (FDR)). The numbers of proteins identified from CF and NCFC mode are 5,483 and 8,529, respectively. This significant increase in the proteome coverage was achieved thanks to the effective fractionation into samples with similar peptide complexities in NCFC mode, and the related increased use of the separation space available in the 2nd RP dimension. By spreading the peptides across the entire LC gradient in the 2nd RP dimension, peptides with low abundance were given a better chance of being detected as MS/MS events. This significantly increased the sensitivity of peptide identification by LC-MS/MS. Interestingly, we observed that the highest number of peptides identified in a CF fraction was lower than the number of peptides identified in the least productive NCFC fraction, suggesting effective dispersal of abundant peptides across fractions, which effectively improved the experimental dynamic range.

Figure 4. Online NCFC allows highly reproducible fractionation. (A) Average numbers of peptides identified in each fraction from triplicate online 2D-NCFC-RP/RP-MS/MS experiments. The error bars indicate the standard deviation in the number of peptides identified. (B) Venn diagram showing overlap between peptides identified in the three experiments. (C) Reproducibility of online NCFC fractionation. Concordance of fraction IDs for the peptides identified in all three experiments. Peptides identified in the same fraction in all three experiments (Red), for two of the three experiments (Grey), and peptides identified in different fractions for all three experiments (Purple).

Reproducibility of fractionation by online NCFC. The reproducibility of the online 2D-NCFC-RP/RPLC system was investigated by repeating the 2D-NCFC-RP/RP-MS/MS experiment three times (Figure 4). In terms of the numbers of peptides identified in each fraction, an average CV of 8.17% was found (Figure 4A). Despite the complexity of the sample analyzed (total tryptic digest of a gastric cancer section) and the under-sampling evident in most LC-MS/MS analyses, the three experiments produced similar numbers of peptide IDs for all

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10 fractions. Obviously, basing our comparison on numbers alone would be misleading, but comparison based on peptide sequence information also revealed very good reproducibility, with a total of 150,949 non-redundant peptides identified in the triplicate experiments, of which 84,287 peptides were identified in all three experiments (Figure 4B). Figure 4C shows how reproducibly the peptides identified in all three experiments were distributed across the 10 fractions of the first dimension. Ca. 99.7% of the common peptides were identified in the same fractions in at least two of the three online 2DNCFC-RP/RPLC replicate experiments, indicating that RPLC combined with NCFC in the 1st dimension is highly reproducible.

CONCLUSIONS A novel online two-dimensional reverse phase/reverse phase liquid chromatography (2D-NCFC-RP/RPLC) separation platform, based on mid-pH reverse phase LC for the 1st dimension and low-pH reverse phase LC for the 2nd dimension, was developed in search of a system allowing comprehensive proteome coverage. The aim was to produce effective fractionation in the first dimension, yielding fractions of similar complexity which could optimally exploit the analytical space of the second separation dimension. The system developed employed an NCFC device based on two ten-position selector valves in the 1st dimension of separation. High duty-cycle and full automation were achieved by connecting the NCFC-RPLC online to a dual-online ultra-high pressure reverse phase liquid chromatography (DO-UHP-RPLC) system. This system consisted of two RP columns (150 cm × 75 µm) and two SPE columns to allow sequential online desalting LC-MS/MS analysis of the fractions produced by the first dimension. With this setup, the fully automated online 2D-NCFC-RP/RPLC system circumvents a number of the labor-intensive and sample-consuming manual processes required with offline 2D-NCFC-RP/RP (offline fractionation, manual pooling, clean-up, drying/reconstitution, and autosampler injection). Thanks to these improvements, sample losses were minimized and reproducibility was high. The online 2D-NCFC-RP/RPLC allowed excellent exploitation of the separation spaces available in the two RPLC stages of the process, resulting in significantly increased peptide identification compared to a contiguous 2DRP/RPLC method. The online 2D-NCFC-RP/RPLC system should therefore allow highly sensitive targeted MS/MS experiments with excellent proteome coverage for samples which require methods to reduce matrix complexity33 while minimizing sample loss compared with offline methods. Also, if two NCFC fractionators (i.e. using two sets of two interconnected 10 position selector valves) were used in parallel to the mRP column, up to 20 NCFC fractions can be generated, which will further increase the proteome profiling coverage of the online 2D-NCFC-RP/RPLC system.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:**

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Fax: 82-2-3290-3121


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Notes

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The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Multi-omics Research Program (NRF-2012M3A9B9036675), Brain Research Program (NRF2014M3C7A1046047), and International Research & Development Program (NRF-2015K1A3A1A21000273) funded by the Korean Ministry of Science, ICT & Future Planning. SL acknowledges LG Yonam Foundation.

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Efficient Exploitation of Separation Space in Two-Dimensional Liquid

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