Automation of nanoflow liquid chromatography ... - Wiley Online Library

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DOI 10.1002/pmic.200600661

Proteomics 2007, 7, 528–539

RESEARCH ARTICLE

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column Xiaogang Jiang1, 2, Shun Feng1, Ruijun Tian1, Guanghui Han1, Xinning Jiang1, Mingliang Ye1 and Hanfa Zou1* 1

National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 2 School of Medicine, Suzhou University, Suzhou, Jiangsu, China

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (mLC-MS/ MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 mm id62 cm SCX trap column and a 75 mm id612 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current mLC-MS/MS system. This system represented a promising platform for routine proteome analysis.

Received: August 30, 2006 Revised: November 21, 2006 Accepted: November 28, 2006

Keywords: Automation / mLC-MS/MS / Sample injection / SCX trap column

1

Introduction

MS is a reliable and highly sensitive technique for protein identification and characterization in proteomics [1, 2]. Prior to MS analysis, complex peptide mixtures are required to be separated effectively. As an orthogonal highly resolving separation technique, 2-DE has firmly held a central place in proteome research [3]. 2-DE of proteins followed with protein identification for the gel spots is still one of the most widely Correspondence: Professor Hanfa Zou, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China E-mail: [email protected] Fax: 186-411-84379620 Abbreviations: FPR, false positive rate; ìLC-MS/MS, nanoflow LC-MS/MS; SCX, strong cation exchange

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

used techniques in the research field of proteomics [4]. Protein in the gel spots is subjected to in-gel tryptic digestion and then identified by either MALDI-TOF MS or HPLC-MS/ MS. Another approach, i.e. the shotgun approach, is to directly analyze complex peptide mixtures resulting from tryptic digestion of complex protein mixtures by HPLC-MS/ MS. Both approaches use HPLC-MS/MS. As a powerful proteome analysis platform, HPLC-MS/MS has made tremendous developments in recent years. Since the introduction of nanoelectrospray source in 1994 [5–11], nanoflow HPLC coupled with MS/MS (mHPLC-MS/MS) has been successfully applied to proteome research which provides dramatic improvement in sensitivity. The main reason of the gain in sensitivity attained by using mHPLC-MS/MS is that * Additional corresponding author: Dr. Mingliang Ye, E-mail: [email protected]

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electrospray mass spectrometer is a concentration-dependent detector. The smaller the id of the analytical column, the higher the concentration of analyte due to the reduced flow rate, thus leading to a higher detection sensitivity. Therefore, in order to achieve a highly sensitive detection, an HPLC column with an extremely small id is preferable. Although C18 packed column with id of 20 mm was successfully applied to proteome analysis [12], columns with id of 75 or 100 mm are mostly used because of their robustness and convenient operation. Although the advantages of mHPLC-MS/MS are apparent, automation of sample introduction onto analytical column remains a challenge in nanoflow HPLC. In typical cases, proteomic sample size ranges from a few microliters to 100 mL. Due to the nanoliter flow rate adopted for the separation of samples on analytical column, it will take a long time if the sample is loaded directly onto the analytical column. Therefore, in order to reduce the sample loading time, a short and larger id trap column may be used. A peptide sample of large volume is first loaded onto the trap column at high flow rate in a short time and then the trapped peptides are eluted from the trap column to an RP analytical column. The automation of this system can be easily realized by directly connecting the trap column with a switching valve [13–17]. However, even using a nanoflow switching valve, dead volume attributed to the valve and transfer lines were still significant which degraded the performance of the chromatography separation. Therefore, the capillary LC system with low dead volume should be developed to improve the separation performance. Licklider et al. [6] reported a fancy vented column system where trap column and analytical column were directly connected via a microcross with an open/close switching valve. The dead volume significantly decreased due to packing of C18 particles into the open space of the microcross. Since the mobile phase for nanoflow HPLC separation did not pass through the switching valve, its channels did not contribute to the dead volume, and therefore instead of using nanoflow switching valve, a regular six-port switching valve could be used. However, the trap column and analytical column could not be replaced independently because there was no frit in the trap column. A similar system was also reported using a custom-made butt tee connector between the trap column and the analytical column [18]. Afterwards, Yi et al. [7] described a modular trap column system in which an independent trap column was connected to an analytical column by a microcross. The trap column and the analytical column were prepared individually and therefore could be replaced separately which would increase the flexibility of the system. An RP trap column coupled with an RP mHPLC-MS/MS was used in all of these reported automated injection systems. In order to improve the separation performance, dead volume between the trap column and the analytical column must be kept very small. This is very challenging for nano-LC system. A good approach is to develop a new column system which can tolerate big dead volume. Using a strong cation © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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exchange (SCX) trap column instead of a C18 trap column may provide this feature since the peptides on SCX trap column can be eluted with hydrophilic buffer and retained on the column head of C18 analytical column before the gradient starts. In this study, we evaluated the use of an SCX trap column to realize the automation of sample injection for mHPLC-MS/MS. The same configuration as reported by Yi et al. [7] was adopted in our automated injection system. The only difference was that the C18 trap column was replaced by an SCX trap column. Sample peptides were first bound onto the SCX trap column at high flow rate, and then they were eluted onto a 75 mm id analytical column by a high salt buffer. The peptides bound on the head of analytical column were finally separated and analyzed when a binary gradient was started. This system was fully automated and no manual intervention was involved. It was found that higher peak capacity could be achieved in the system using SCX trap column than that using C18 trap column. Superior proteomics coverage was demonstrated by the analysis of tryptic digest of BSA and yeast cell lysate. This system could also be very conveniently applied to multidimensional separation of complex peptide mixtures.

2

Materials and methods

2.1 Materials Magic C18 AQ (5 mm, 100 and 200 Å pore) was purchased from Michrom BioResources (Auburn, CA, USA), and polysulfoethyl aspartamide (5 mm, 200 Å pore) was from PolyLC (Columbia, MD, USA). PEEK tubing, sleeves, microtee, and microcross were obtained from Upchurch Scientific (Oak Harbor, WA, USA). Fused-silica capillaries (50, 75, and 100 mm id) were purchased from Polymicro Technologies (Phoenix, AZ, USA). Water used in the experiments was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Tetramethoxysilane (TMOS) was obtained from Chemical Factory of Wuhan University (Wuhan, China), and PEG, (Mr = 10 000) was from Aldrich (Milwaukee, WI, USA). DTT, and iodoacetamide were purchased from Sino-American Biotechnology Corporation (Beijing, China). Urea, ammonium acetate, ammonium bicarbonate, and acetic acid were obtained from Sigma (St. Louis, MO, USA). Trypsin was obtained from Promega (Madison, WI, USA), and Tris from Amersco (Solon, Ohio, USA). Formic acid was obtained from Fluka (Buches, Germany), and ACN (HPLC grade) from Merck (Darmstadt, Germany). Human hepatocellular carcinoma cell line SMMC-7721 was a gift from Shanghai Cancer Institute. 2.2 Sample preparation The yeast protein extract was prepared in a denaturing buffer containing 50 mM Tris/HCl (pH 8.1) and 8 M urea as before [19]. The protein concentration was determined by BCA www.proteomics-journal.com

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assay. The protein sample was reduced by DTT and alkylated by iodoacetamide. Then, the solution was diluted to 1 M urea with water, and the pH value was adjusted to 8.1. Finally, trypsin was added (trypsin/protein, 1:50) and incubated at 377C for 20 h. The tryptic digest was desalted with a C18 solid-phase cartridge. The tryptic digest of BSA was prepared in the same way. Human hepatocellular carcinoma cell line SMMC-7721 was homogenized in lysis buffer (50 mM Tris, 8 M urea, 4% CHAPS, 65 mM DTT), and then sonicated for 60 s followed by centrifugation at 25 0006g for 1 h. The supernatant was collected as the crude extraction, which was further precipitated overnight with five volumes of a solution containing ethanol, acetone, and acetic acid (50:50:0.1, v/v/v) at –207C. The supernatant was carefully removed after centrifugation at 12 0006g for 1 h at 47C. The precipitated protein sample was redissolved in 50 mM Tris, 8 M urea (pH 8.1), and stored at –207C. Digest of SMMC-7721 protein by trypsin was the same as that of the yeast sample. 2.3 Column preparation Columns were packed using a homemade pneumatic pressure cell at constant nitrogen gas pressure of about 580 psi with a slurry packing method [7]. For the preparation of the analytical column, one end of a 75 mm id fused silica capillary was first manually pulled to a fine point of 5 mm with a flame torch according to the protocol from the Institute of Systems Biology (the protocol is available online at: http:// www.proteomecenter.org/protocols.php). The end of the tip was trimmed by a capillary cutter and checked by microscope. Then, the C18 particles were packed until the packing section reached the length of 12 cm. Fused silica capillary with id of 100 mm was used to prepare the trap column. Prior to packing the resin, a porous frit was prepared to retain resin particles at one end of the capillary by a simplified method basically described by Xie et al. [20, 21]. Briefly, the polymerization mixture containing 1.06 g PEG, 4.5 mL TMOS, and 10 mL of 0.01 M acetic acid was violently agitated to promote hydrolytic reaction under ice-bath for 45 min. A 1–2-mm segment of the resulting solution was then drawn into the capillary, followed by incubation in a thermostated bath at 407C for 24 h. The trap column was finally prepared by packing 2 cm length of ODS or SCX resin into the capillary with frit. 2.4 HPLC and MS The HPLC-MS/MS system consisted of a quaternary Surveyor pump, a Surveyor autosampler, and an LTQ linear IT mass spectrometer equipped with a nanospray source, and a six-port/two-position valve (Thermo, San Jose, CA). The four buffer solutions used for the quaternary pump were 0.1% formic acid (buffer A), 99.9% ACN/0.1% formic acid (buffer B), 500 mM NH4Ac/5% ACN, pH 3 (buffer C), and 5% ACN/ 0.1% formic acid (buffer D). The temperature of the ion© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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transfer capillary was set at 2007C. The spray voltage was set at 1.8 V and the normalized collision energy was set at 35.0%. An automated gain control function was used to manage the number of ions injected into the IT. One microscan was set for each MS and MS/MS scan. All MS and MS/ MS spectra were acquired in the data-dependent mode. The mass spectrometer was set such that one full MS scan was followed by seven MS/MS scans on the seven most intense ions. The dynamic exclusion function was set as follows: repeat count two, repeat duration 30 s, and exclusion duration 90 s. All the procedures, including sample loading, valve switching, gradient elution, and data collection, were fully automated and controlled by the Xcalibur software version 1.4 (Thermo). 2.5 Automated injection system The configuration for automated sample injection system using SCX trap column was very similar to that of using C18 trap column reported by Yi et al. [7]. As shown in Fig. 1, this system was composed of an autosampler, a six-port/twoposition switching valve, a microtee, and a microcross. The SCX trap column was connected to a C18 analytical column by a microcross. The ESI voltage was supplied through the platinum wire plugged in the microcross. The six-port/twoposition switching valve directed eluting flow from the trap column to waste or the analytical column. The procedure of automated sample injection followed with mHPLC-MS/MS analysis using the SCX trap column is also shown in Fig. 1. There were three steps: (i) Loading the sample onto the SCX trap column. The sample (20 mL) was injected by Surveyor autosampler using the no-waste injection mode at a flow rate of 2–5 mL/min with the switching valve in position A. The mobile phase used was 0.1% formic acid containing 5% ACN. During the sample-loading step, the split flow at the front of the trap column was closed and the flow through from the SCX trap column was directed to waste. (ii) Eluting the sample from the SCX trap column onto C18 analytical column. Once the sample has been loaded, the valve was switched to position B activating the flow splitting at the front of the trap column and directing the flow to the analytical column. The flow rate to the analytical column was about 200 nL/min after splitting. The peptides retained on SCX trap column were eluted onto the analytical column by flushing with a buffer containing 500 mM NH4Ac/5% ACN, pH 3 (buffer C) for 5 min. (iii) Gradient separation. After the system was equilibrated with buffer A for 5 min, a binary gradient with buffer A and buffer B was started for the separation. For the analysis of tryptic digest of BSA, a gradient was developed from 5 to 35% buffer B for 50 min, and from 35 to 80% for 10 min. For the analysis of tryptic digest of 1 mg yeast protein, a binary solvent composition gradient with buffer A and B was developed from 5 to 35% buffer B for 110 min, and from 35 to 80% for 15 min. For the system using C18 trap column, www.proteomics-journal.com

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Figure 1. Schematic diagrams of sample injection systems: (A) SCX trap column system and (B) a manual injection system.

the procedure was similar. The only difference was that the salt elution (step 2) was omitted. After the sample was loaded onto the C18 trap column, a gradient directly started for the separation. For the manual sample loading, an open capillary filled with sample was connected between the microcross and the analytical column. The peptide sample in the open capillary was flushed onto the analytical column by the mobile phase A and retained on the head of the analytical column. After the open capillary was removed, the gradient elution was then started as described above. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.6 Multidimensional separation using the system with SCX trap column The automated nanoflow HPLC-MS/MS system using SCX trap column was also used for multidimensional separation. Tryptic digest of 18 mg of SMMC-7721 protein was loaded onto the SCX trap column. Then, a series of salt elution steps with salt concentrations of 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, and 300 mM NH4Ac were used to elute peptides from the trap column onto the analytical column. The salt bumps were automatically generated by the quaternary www.proteomics-journal.com

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pump using buffer C and D. After each salt elution, the two columns were equilibrated for 5 min with buffer A. Then the peptides retained on the analytical column were separated by ramping a gradient from 5 to 35% buffer B for 110 min, 35 to 80% for 15 min. After the column was flushed by 80% of buffer B for 10 min and equilibrated with 5% of buffer B for 15 min, a new cycle started by another peptide fraction displaced with a higher salt concentration from the SCX trap column. For the last two cycles, the column was washed with 100% buffer C for 20 min in order to elute all peptides from the SCX trap column. The RP gradient described above for yeast protein digest was applied to each multidimensional HPLC separation cycle to separate peptides prior to MS detection. In this experiment, 14 cycles were performed for the entire analysis. 2.7 Protein identifications The acquired MS/MS spectra were then used to perform database searching using the TurboSEQUEST in the BioWorks 3.2 software suite (Thermo). The yeast and BSA composite database were used for the search of data from BSA and yeast samples, and Human International Protein Index (IPI) protein sequence database (v3.04) was used for that of SMMC-7721 sample. Reversed sequences were appended to both databases for the evaluation of the false positive rate (FPR). Cysteine residues were searched as a static modification of 57.0215 Da, and methionine residues as a variable modification of 115.9949 Da. Peptides were searched using fully tryptic cleavage constraints and up to two missed internal cleavages sites were allowed for tryptic digestion. The mass tolerances were 2 Da for parent masses and 1 Da for fragment masses. The peptides were considered as positive identification if Xcorr were higher than 1.9 for singly charged peptide, 2.2 for doubly charged peptide, and 3.75 for triply charged peptides, and DCn cut-off values were 0.19. FPRs were calculated by using the following equation, FPR = 26n(rev)/(n(rev) 1 n(forw)), where n(forw) and n(rev) are the number of peptides identified in proteins with forward (normal) and reversed sequence, respectively [22, 23]. FPR less than 5% was obtained for the peptide identifications by using the above parameters. If not otherwise stated, the proteins were identified based on one peptide minimum.

3

Results

3.1 SCX trap column system vs. C18 trap column system Introduction of dead volume is an inevitable problem in the application of trap column, which will typically result in gradient profile distortion and peak broadening in the nanoflow HPLC. The automated injection system may also results in sample loss and contamination due to system carry over and © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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peptide adsorption on the tubes of the system. Therefore, the degradation of the performance of proteome analysis using automated sample injection can occur. To date, the performance of the developed automated injection system has been validated only by running a few proteome samples. The compromise to the separation performance resulting from automation has not been investigated yet. An ideally automated system for sample injection should allow samples to be loaded in a short time, in the meanwhile, only a limited compromise to the separation performance occurred. In this study, in order to evaluate the compromise, the performance of the automated injection systems using an SCX trap column and a C18 trap column were compared with that of a manual injection system. Manual injection of a sample onto a nanoscale HPLC column in proteome analysis is typically achieved using a pneumatic pressure cell [7]. In this study, a sample, typically 2–5 mL, was placed in a glass vial and sealed in the pressure cell. The capillary analytical column was removed from the HPLC pipeline and its entrance end inserted into the pressure cell. The sample was injected onto the column using gas pressure. The amount of sample injected was monitored by measuring the volume of solution eluted from the column tip. After loading the sample, the capillary column was reconnected to the HPLC pipeline. This approach was simple and practical; however, the injected volume could not be accurately measured. In order to accurately control the amount of injected sample, sample loading was achieved manually by using a sample capillary [19] (Fig. 1B). An open capillary with volume of 1 mL was filled with sample solution, and then it was connected between the microcross and the analytical column in the HPLC pipeline. The peptide sample in the open capillary was flushed onto the analytical column with buffer A and retained on the column entrance end. After loading the sample, the sample capillary was removed. In this approach, the amount of sample loaded could be accurately controlled by the volume of the capillary. Since the sample was directly loaded onto the analytical column, no the sample loss occurred in the manual injection. Compared with the automated injection systems using a trap column, better performance of the separation could be achieved with manual injection since no extra volume was introduced to the nanoflow HPLC system. Therefore, manual injection represents the ideal injection mode in terms of separation. Its performance was used as a standard to evaluate the performance of the automated injection systems. Tryptic digest of 1 pmol of BSA was selected as a test sample to investigate the performance of the separation obtained in the three systems, i.e. systems with manual injection, automated injection systems using a C18 trap column, and that using an SCX trap column. Three replicate runs were conducted for each case and the obtained typical base peak chromatograms are shown in Figs. 2(A–C), respectively. Serious delay of the gradient was observed for the C18 trap column system. More importantly, its separation window decreased dramatically. The separation window www.proteomics-journal.com

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separation window was not observed for the automated injection with an SCX trap column. Only a slight delay in elution time was observed. Furthermore, the peak with an SCX trap column system was sharper than that with a C18 trap column. As an example for the two systems, the extracted ion chromatogram of a typical peptide (K.HLVDEPQNLIKQNCDQFEK.L) with m/z 786 can be seen in Supplementary Figs. 1(A) and (B). The peak width at half height for the C18 trap column system was about 40 s, while that for the SCX trap column was only 14 s. Because of wider separation window and sharper peak, the SCX trap column system had higher peak capacity for the separation of peptides and so more peptides should be identified. After database searching, as shown in Table 1, 91 unique peptides from BSA were identified resulting in sequence coverage of 90% when the SCX trap column was used. If BSA is digested by trypsin without missing cleavage sites, it will generate 55 unique peptides with MW.500 Da. But more than 55 unique peptides were identified from BSA in this study. This implied a large number of identified peptides containing missed cleavage sites. The number of identified peptides and the BSA sequence coverage obtained for the SCX trap column were very similar to that of 92 unique peptides and 91% of sequence coverage obtained with manual injection. However, for the C18 trap column system only 54 unique peptides and sequence coverage of 77% were obtained. The peptides identified from both systems are listed in Supplementary Table 1. The use of the SCX trap column system led to the identification of more unique peptides. These results indicate that the performance of the SCX trap column system is better than its equivalent C18 trap column system. The performances of these three systems were further investigated by analyzing a more complex sample, tryptic digest of yeast lysate. The typical base peak chromatograms for the separation of a tryptic digest of 1 mg yeast protein are shown in Fig. 3. After database searching, the average of identified unique peptides of 1641, 889, and 1471 were obtained for three replicate runs by using a manual injection system, a C18 trap column system and an SCX trap column system, respectively. As shown in Table 1, the corresponding Table 1. Comparison of the performance of different injection approaches: (A) manual injection; (B) automated injection using a C18 trap column, and (C) automated injection using an SCX trap column

A Figure 2. Base peak chromatograms of tryptic digest of BSA obtained with (A) manual injection and automated injection using (B) C18 trap column, (C) SCX trap column. Peptides were separated using a 50-min gradient from 5 to 35% buffer B (buffer B: 99.9% ACN/0.1% formic acid).

of about 37 min was obtained for manual injection, while the window decreased to about 27 min for the automated injection with a C18 trap column. The obvious decrease in the © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

B

C

Analysis of 1 pmol of tryptic digest of BSA (based on three replicate runs) Number of unique peptides 92 6 2 54 6 1 91 6 3 Sequence coverage (%) 91 6 1.5 77 6 0.6 90 6 0.6 Analysis of 1 mg of tryptic digest of yeast lysate (based on three replicate runs) Number of unique peptides 1641 6 40 889 6 62 1471 6 51 Number of identified proteins 527 6 2 339 6 10 451 6 13


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Figure 3. Base peak chromatograms of tryptic digest of 1 mg of yeast protein obtained with (A) manual injection and automated injection with (B) C18 trap column, (C) SCX trap column. Peptides were separated using a 110-min gradient from 5 to 35% buffer B (buffer B: 99.9% ACN/0.1% formic acid).

identified protein numbers were 527, 339, and 451, respectively. The compromise of protein identifications was obvious for both the automated injection systems. But the number of identified peptides decreased only by 9% with the SCX trap column system while the number for the C18 trap column dramatically decreased by 44%. Clearly, superior proteomics coverage could be obtained for the SCX trap column system. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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The dead volume resulted from the frit of the trap column and the open space in the microcross between the trap column and the analytical column. Since the same configuration was used here, the involved dead volumes were the same for both systems, but significant degradation of the separation was only observed for the C18 trap column system. It can be seen from Figs. 2 and 3 that a significant delay was observed for the C18 trap column system. The delay of more than 20 min was also found in the chromatograms in previous reports [6, 7], where a vented column system and C18 trap column were used for automated sample injection for nano-LC-MS/MS analysis. It was interesting that a significant delay was not observed for the SCX trap column system. This is unexpected since the C18 trap has identical dimensions and is located at the same position as the SCX column. The systems were run multiple times and reproducible results have been obtained. This may be explained by the different separation behaviors of analytes in these two trap column systems. For the C18 trap column system, a gradient directly started after the peptides were loaded onto the trap column. Thus, peptides were gradually eluted from the trap column, stayed in the open space in the microcross for a while and then gradually bound onto the analytical column again during the RPLC gradient. For this case, the C18 trap column and C18 analytical column were coupled in tandem and the separation of peptides was achieved across the two columns. It could be observed from Fig. 2 that a significant decrease in the separation window and broadening of the peaks had occured. Obviously, the separation performance of the C18 trap column system was not good. However, for the SCX trap column system, the peptides bound on the trap column were first eluted onto the analytical column by 500 mM NH4Ac buffer, and then peptides were retained and concentrated on the entrance end of the analytical column under this hydrophilic buffer. Therefore, these peptides were only separated on the C18 analytical column by starting a gradient. It was found that the serious decrease in the separation window and obvious degradation of the separation observed on the C18 trap column system were not observed for the SCX trap column system. The process in the SCX trap column system was very similar to that of manual injection system where the sample was also retained at the entrance end of the analytical column before the gradient started. Therefore, similar separation window and peak capacity were obtained for both of these two systems. However, the elution profiles were very different for these two systems (Fig. 2A vs. Fig. 2C and Fig. 3A vs. Fig. 3C). This was probably caused by the disturbance of gradient profile when the mobile phase flowed through the SCX trap column and the cross. The use of the SCX trap column might also result in the loss of some peptides which led to the change in elution profile. Since only a slight decrease in the number of identified peptides and proteins was found with the SCX trap column system compared with that of manual injection, the loss of peptides during sample loading was not significant. www.proteomics-journal.com

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The procedure of running the SCX trap column system is shown in Fig. 1A. Compared with the C18 trap column system, an extra step of salt elution was used to transfer the peptides from the trap column to the analytical column in the SCX trap column system. Since the salt solution was directly introduced into the C18 analytical column, it is important to use a volatile buffer to prevent the clogging of the analytical column and suppression of peptide ionization. NH4Ac buffer was used to generate salt bumps for 2-D separation using a biphasic column [24]. NH4Ac buffer with a concentration of up to 500 mM was directly introduced into the analytical column which was also coupled directly to a mass spectrometer. Compromise on the separation and detection of peptides was not observed in their study. Thus, NH4Ac buffer was also selected to elute peptides from SCX trap column to the C18 analytical column in our case. The clogging of nanospray emitter and suppression of peptide ionization were not observed. The salt elution step spent only 5 min and no extra equilibration time was required. During the loading sample procedure, the flow through from the SCX trap column was directed to waste. This design of the trap column system enabled a sample to be loaded at high flow rate with low back pressure. Flow rate of 2 mL/min was adopted to load the sample onto the trap column, which was ten times higher than that of the flow rate in the analytical column.

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3.2 Performance of the SCX trap column system To be an automated mHPLC-MS/MS system for routine proteome analysis, the system must be very reproducible and sensitive. Nine consecutive analyses of a tryptic digest of 1 pmol of BSA were conducted to evaluate the reproducibility of the automated injection system using an SCX trap column. Pep3D [25], which displayed LC-ESI-MS data in a 2-D (retention time vs. m/z) density plot, was used to visualize the obtained data. Very similar patterns of Pep3D images were obtained as the two typical images were shown in Fig. 4, which indicated good repeatability of this system. In order to quantify the reproducibility of the system, the retention times of five identified peptides with different m/z and retention times were extracted from the Pep3D images labeled as A to E. The results are listed in Table 2. The values of RSD of less than 1.01% were obtained for the five peptides with the nine consecutive runs. The reproducibility of peptide intensity across replicate runs was also checked and the results are listed in Table 2. It can be seen that the signal intensity of the peptide had no significant change. In addition, the number of unique identified peptides (91 6 2) and the resulting BSA sequence coverage (90 6 1%) were all similar for these LC-MS/MS runs. Besides the BSA digest, the analysis of a more complex sample, the tryptic digest of yeast lysate, was also performed to evaluate its reproducibil-

Figure 4. Pep 3-D images for the analysis of tryptic digest of 1 pmol of BSA by the SCX trap column system. (a) Run 3 and (b) run 9. Indicated peptides: A, R.FKDLGEEHFK.G (m/z 625); B, R.RHPEYAVSVLLR.L (m/z 721); C, R.KVPQVSTPTLVEVSR.S (m/z 821); D, K.CCAADDKEACFAVEGPK.L (m/z 965); E, K.SHCIAEVEKDAIPENLPPLTADFAEDK DVCK.N (m/z 1172).



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Table 2. Reproducibility for separation of tryptic digest of BSA by the SCX trap column system (n = 9)

Peptidea)

A

B

C

D

E

Retention time (min) Peak intensity (107)

17.13 6 0.15 0.143 6 0.04

26.35 6 0.21 1.30 6 0.17

24.60 6 0.25 1.62 6 0.27

38.76 6 0.27 0.141 6 0.04

35.69 6 0.27 0.138 6 0.04

a) Refer to Fig. 4 for the peptide sequences and their locations in Pep3D images.

ity. The three replicate runs resulted in the identification of 1417, 1519, 1478 unique peptides corresponding to 442, 466, and 445 yeast proteins, respectively. The number of identified peptides and proteins was also very similar for the yeast sample between different runs. These results demonstrated the good reproducibility of this system. The sensitivity of the system was first evaluated by analyzing different amounts of BSA digest. The obtained results are listed in Supplementary Table 2. At least three separate runs were conducted for each sample. Consistent results were obtained when the amount of the tryptic digest analyzed was above 1 fmol. A similar number of identified peptides and protein sequence coverage was obtained for each replicate run. With a decrease in the injected amount, the BSA protein-sequence coverage also decreased. Sequence coverage of about 46% could still be obtained with injection of tryptic digest of only 1 fmol BSA. With a further decrease in the amount of sample injected, the sequence coverage was dramatically decreased, and the results were less consistent. For example, for the injection of 100 amol digest, the sequence coverage fluctuated from 15 to 25% and the number of identified peptides fluctuated from 7 to 13 for different replicate runs. As a routine proteome analysis platform, consistent results should be obtained. Therefore, the sensitivity of this platform was in the range of high attomole to low femtomole for the analysis of BSA digest. The sensitivity of the system was also evaluated by analysis of different amounts of yeast sample. As shown in Supplementary Table 2, with the decrease in the injecting amount, the number of peptides and proteins identified also decreased. Big fluctuations were observed in the number of identified peptides and proteins for replicate runs when the amount of injected sample was less than 100 ng of the extracted proteins. To obtain consistent results for the analysis of complex proteome samples by this system, at least 100 ng sample should be loaded. The tryptic digest of BSA and yeast protein extract were desalted by a C18 cartridge prior to being loaded onto the SCX trap column, and no significant sample loss was observed. However, if a high concentration of salt was present in the sample, many peptides might not be bound onto the SCX trap column which would result in huge sample loss. Ammonium bicarbonate (NH4HCO3) was often used as the enzymatic digestion buffer in many proteomics researches [11, 22, 26–28]. Therefore, yeast tryptic digest prepared in 50 mM NH4HCO3 was used to evaluate the salt tolerance © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of this system. As we described earlier, the analysis of 1 mg yeast digest without the presence of salt resulted in the identification of 1471 unique peptides and 451 proteins (see Table 1). The same amount of yeast tryptic digest with 50 mM NH4HCO3 was analyzed with the same conditions, 1363 6 57 unique peptides and 400 6 7 proteins were identified for three replicate runs. The number of identified peptides was decreased by 7% which indicated that peptide loss during sample loading was relatively significant in the presence of 50 mM NH4HCO3. Therefore, desalting of the sample was necessary. Since NH4HCO3 was a volatile salt, it might be partially removed by lyophilizing the sample in Speed Vacuum. Yeast tryptic digest (1 mg) containing 50 mM NH4HCO3 was lyophilized and then redissolved in 0.1% formic acid. Identification of 1478 6 47 unique peptides and 441 6 13 proteins was achieved after the analysis of this sample. The average number of identified peptides decreased from 1478 to 1363, if the sample pretreatment by removing volatile salt was not performed. Comparing the number of peptides and proteins identified in both cases, it indicated that sample loss due to Speed Vacuum was not very significant. However, if a higher concentration of salt was present in the sample, use of a C18 cartridge to remove salt was necessary. This would result in sample loss especially for trace-level proteins. Compared with the automated injection system using a C18 trap column, an obvious disadvantage of the SCX trap column system was that an extra step of desalting of the sample prior to the analysis was required if a high concentration of salt was present in the sample. Considering the significant improvement in the separation for the SCX trap column system, the sample pretreatment prior to analysis was worthwhile. 3.3 Application of SCX trap column system in multidimensional separation In recent years, multidimensional separation of peptide mixtures prior to MS analysis was introduced for large-scale proteomics analysis [24, 29, 30]. 2-D separation of peptides could be easily achieved with a biphasic column where both SCX and C18 resins were packed into the same capillary column [24]. After loading the sample, the 2-D separation could be operated in automated mode by using a quaternary pump [28]. In the fully automated injection system developed in our case, two stationary phases, SCX and C18, were also used. It was found that this system could also be conwww.proteomics-journal.com

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veniently adopted for two-dimensional separation of peptides. Two channels of the quaternary pump generated a binary organic solvent (water/ACN) gradient for RP chromatography and two other channels generated a salt step gradient for strong cation-exchange chromatography. Therefore, no extra pump was required for implementation of the 2-D separation. The procedure of loading sample was the same as that of 1-D separation. However, the procedure for elution of peptides from the SCX trap column was different. Instead of using a high salt buffer to elute all bound peptides in 1-D separation, the peptides bound on the SCX trap column were eluted gradually with a series of salt steps. Each peptide fraction from the SCX trap column was transferred to the RP analytical column for LC-MS/MS analysis. The 2-D separation using the SCX column system was applied to the analysis of SMMC-7721 proteome. After loading 18 mg of tryptic digest of SMMC-7721 protein, 14 cycles of salt step elution of peptides from the SCX trap column followed by RP separation of the eluted peptide fractions were automatically conducted. Supplementary Figure 2 shows base peak chromatograms for each cycle. Different patterns were observed for each peptide fraction demonstrating the orthogonal separation of the system. After a database search, a total of 5417 unique peptides and 1906 protein groups were identified. Among 1906 protein groups, 920 protein groups were identified by at least two unique peptides. The results indicated good performance of the SCX trap column system for multiple dimensional separations.

4

Discussion

Development of a fully automated high performance platform for routine proteome analysis is an important task for proteome research. A nanoflow HPLC-MS/MS system represents a highly sensitive and high-throughput platform for proteome analysis. Because of the contradiction of low flow rate and large sample size, the automation of sample injection for the mHPLC-MS/MS system is challenging. Using a trap column prior to the analytical column allows large volumes of sample to be loaded in a short time. The automation could be achieved by using either a fancy vented column system [6] or directly using a nanoflow switching valve [7]. RP C18 trap columns were used for all of these systems. However, the performance of the automated injection system using the C18 trap column was not good. According to the results of our study, the delay of elution time and broadening of peaks were observed for this system. This seriously resulted in a decrease in peak capacity and therefore the number of identified unique peptides decreased by about 44% compared with that of the manual injection system. In order to improve the performance of the automated proteome analysis platform, an SCX trap column was applied to realize the automation of sample injection for the first time. It was found that the situation was significantly improved when the C18 trap column was © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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replaced by an SCX trap column. Decrease in peak capacity was not observed in the SCX trap column system. Compared with that of the manual injection system, the number of identified peptides decreased only by about 9%, which was much lower than that of the C18 trap column system. For an SCX trap column system, peptides are refocused on the C18 analytical column when an aqueous buffer of low organic solvent is used to elute peptides from the SCX trap column. While for the C18 trap column system, refocusing of peptides onto the C18 analytical column with organic containing buffer, which is used for elution of peptides from the C18 trap, is not as effective when compared to the SCX trap setup, and therefore the chromatographic separation is compromised. An ideal automated sample injection system should allow samples to be loaded in a short time, with limited compromise to the separation performance. As we demonstrated before, no significant degradation in the separation was observed in an SCX trap column system. As for the injection time, extra 5 min for salt elution was required to transfer peptides from the SCX trap column to the analytical column. Considering the significant improvement in proteome analysis, it was worthwhile to spend more time on injection. In this study, 2 mL/min of flow rate was adopted which enabled loading of a 20 mL sample onto the SCX trap column in 10 min. The dimensions of the SCX trap column were 100 mm id62 cm. In addition, a bigger id trap column could also be used which allowed using a higher flow rate and, therefore, further decreased the loading time. This automated mHPLC-MS/ MS system consisted of a microtee, a microcross, an autosampler, a quaternary pump, and an LTQ linear IT mass spectrometer equipped with a six-port switching valve. Except for the microtee and microcross all the other components were from the standard setup of a commercial LCMS/MS system. The automation of this system was controlled by the Xcalibur software. No extra hardware and software were required for this system, which facilitated the use of this system in other laboratories. Since an SCX trap column was used, one concern about this platform was its salt tolerance. As we had investigated, the presence of 50 mM NH4HCO3 in the sample resulted in a decrease in the number of identified peptides by 7%. Similar to SCX analytical column, the salt concentration in the sample should be less than 20 mM [31]. Protein extract was typically prepared in a denaturing solution with about 100 mM buffer. Because trypsin only tolerates low concentration of denaturant like urea, the protein extract need to be diluted by four to eight folds before the addition of trypsin. After dilution, the salt concentration in the resulting enzymatic digest was often less than 20 mM [4, 22, 31–33]. Thus, majority of proteome samples could be directly submitted to the SCX trap column system for automated analysis without any pretreatment. This system was a promising platform for routine proteome analysis because of its high performance. However, desalting prior to analysis was required in the www.proteomics-journal.com

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presence of high concentration of salt. Majority of the volatile salt may be removed by drying down in Speed Vacuum. But to remove nonvolatile salt, use of C18 solid-phase cartridge should be a good choice. Compared with C18 trap column system, an extra step of sample pretreatment was required for an SCX trap column system, which made this approach more labor intensive. However, considering the low compromise to separation, it was worthwhile to use the automated proteome analysis system using an SCX trap column. After the salt elution step, there was an equilibrated process with 0.1% formic acid/5% ACN. As a result, there should be almost no salt in the electrospray droplets when the gradient started. Therefore, the application of this technique could be extended to couple any other ESI-based mass spectrometers besides IT MS. Multidimensional chromatography coupled to MS/MS is a powerful platform for large-scale proteome analysis. Multidimensional chromatography is most commonly accomplished by the combination of SCX and RP chromatography because of their orthogonal separation properties. In addition to performing automated 1-D separation, the automated sample injection system using the SCX trap column could be very conveniently employed for online multidimensional separation of peptides without any modification. The peptides bound on the SCX trap column were gradually eluted onto the RP analytical column by salt step gradients which were generated by two channels of the quaternary pump, and then each peptide fraction was subjected to an RP chromatography separation. A total of 5417 unique peptides and 1906 proteins were identified from an SMMC-7721 proteome sample after 14 cycles of running using the SCX trap column system in this study. 2-D separation of peptides could also be achieved with biphasic column where both C18 and SCX resins were packed into the same capillary [24]. The automation of the separation procedures in biphasic column was similar to that of the SCX trap column system in this study. However, manual sample introduction was required for the biphasic column approach if it was operated in nanoliter flow rate. The sample introduction was typically achieved by using a pressurized cell. This procedure was labor intensive and sometimes the column might be clogged during the sample loading step. In contrast, the trap column structure with the SCX trap column approach enabled the mobile phase to go through the SCX trap column at high flow rate during sample loading step. Thus, the sample could be automatically loaded in a short time. Higher sample capacity could be achieved for the SCX trap column system due to the use of an SCX trap column with bigger id. The significant advantage of using an SCX trap column system for 2-D separation was the complete automation of this system. The sample was loaded onto the SCX trap column and 2 days later the analysis was finished. No manual intervention was involved. The SCX trap column system represented a promising online multidimensional separation platform for large-scale proteome analysis. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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5

Conclusions

In order to improve the separation performance of automated injection system using C18 trap column, it is crucial to minimize the introduced dead volume resulting from the trap column and related connections. For nanoflow HPLC system, further reduction in the dead volume between trap column and analytical column is mechanically difficult. A different choice of approach is to develop an alternative system which can reduce the sensitivity to dead volume. In this paper, we have reported for the first time a new approach using an SCX trap column to automate the sample introduction step for nanoflow LC-MS/MS analysis. The construction of this system was easy and required few changes in the current nanoflow LC-MS/MS system. The modular design of this trap column system enabled the flexibility of replacing the SCX trap column and the C18 analytical column separately when it was necessary. This system was fully automated and easy to maintain. As we have demonstrated, this system could be conveniently applied to two- as well as one-dimensional separation of peptides. The SCX trap column system allowed the sample to be loaded in a short time and, in the mean time, maintained a high separation performance. This system has reduced the sensitivity to dead volume when compared to the C18 trap and was a good alternative for the automated system previously using the C18 trap column for routine proteome analysis.

Financial supports from the National Natural Sciences Foundation of China (20327002, 20675081), the China State Key Basic Research Program Grant (2005CB522701), and the Knowledge Innovation program of DICP to H. Z. and National Natural Sciences Foundation of China (No. 20605022) to M. Y. are gratefully acknowledged.

6

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