MS Separations To

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Anal. Chem. 2004, 76, 5180-5185

Optimization of Two-Dimensional Off-Line LC/MS Separations To Improve Resolution of Complex Proteomic Samples Martin Vollmer,* Patric Ho 1 rth, and Edgar Na 1 gele

Agilent Technologies R&D and Marketing GmbH and CoKG, Hewlett-Packard-Strasse 8, D-76337 Waldbronn, Germany

In off-line 2D-HPLC a continuous salt gradient is applied in the first separation dimension. This increases the number of identified proteins from complex samples significantly due to higher chromatographic resolution compared to stepwise elution. Achievement of optimal resolution requires the optimization of the two separation dimensions. The influence of LC elution gradients in the first and second dimensions, of analysis time, of stationary-phase material, and of column dimensions was systematically investigated in order to obtain information on the overall peak capacity of the separation system. Provided data indicate that for complex samples such as an E. coli cell extract, a shallow LC SCX gradient with a high number of collected fractions significantly increases the overal peak capacity while for lower complexity samples short gradients with few fractions were sufficient to obtain a maximum of identified peptides. In addition, column dimensions and materials exihibited a strong effect on the overall efficiency of the 2D HPLC separation. The outcome of these experiments could hence serve as a guideline for investigators to adapt their method for the separation of their specific proteome sample to achieve a maximum of peptide sequence information by 2D LC MS/MS analysis. Proteomics was first defined by Wilkins as the study of the protein complement of the genome.1 The scope of profiling proteomics is to identify a complete set of proteins and its modifications from a cell, a body fluid, or a subcellular fraction expressed at any one time.2 In contrast to mRNA expression analysis, proteomic profiling indicates the actual rather than the potential functional state of a cell or a tissue.3 Proteomic approaches will finally foster a better understanding of disease pathogenesis and push the development of new disease markers and early disease diagnostics, as well as accelerate new drug development.4 * To whom correspondence should be addressed. E-mail: martin_vollmer@ agilent.com. Fax: ++ 49 (7243)602414. (1) Wasinger, V. C.; Cordwell, S. J.; Cerpa-Poljak, A.; Yan, J. X.; Gooley, A. A.; Wilkins, M. R.; Duncan, M. W.; Harris, R.; Williams, K. L.; Humphery-Smith, I. Electrophoresis 1995, 16, 1090-1094. (2) Simpson, R. J. Proteins and Proteomics: A Laboratory Manual, 1st ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2003. (3) Wasinger, V. C.; Corthals, G. L. J. Chromatogr., B 2002, 771, 33-48. (4) Hanash, S. Nature 2003, 422, 226-232.

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The analysis and identification of proteins in the background of highly complex proteomic samples with 104-105 different protein species and a dynamic range from 106 in cells5 and up to 1010 in blood6 demand analytical techniques with tremendous resolving power. Since by far no available single-separation technology is currently able to cope with these requirements, comprehensive proteome analyses require a smart combination of sample preparation, prefractionation, labeling techniques, affinity selection, and high resolving separation techniques prior to detection by MS and MS/MS. By using nanoelectrospray MS/MS detection, it is especially crucial to consider duty cycles of the MS instrument to provide the MS enough time for the successful identification of all eluting peptides. This means in order to identify even low-abundance proteins, qualitative and quantitative changes, and posttranslational modifications, the proteome sample entering the MS instrument has to be reduced dramatically in complexity. Following prefractionation or affinity selection techniques, two different high-resolution approaches have been developed to address this ambitious goal. These are 2D gel electrophoresis (2DGE)7,8 and two- or multidimensional HPLC (2DLC).9-11 While 2DGE is currently unsurpassed in resolution, it still suffers from some important drawbacks that can be overcome by using liquidphase separations. Automation of 2DGE is hardly achievable, and hydrophobic membrane proteins, basic and acidic proteins, and very large and small proteins are poorly resolved or recovered.12 Since 25% of the different cell proteins are membrane-associated proteins, but in contrast, 75% of all drug targets belong to this protein class, the 2DLC technology provides an excellent alternative to especially address these protein subspecies.13 (5) Tyers, M.; Mann, M. Nature 2003, 422, 193-197. (6) Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics 2002, 1.11, 845867. (7) O’Farrel, P. H. J. Biol. Chem. 1975, 250, 4007. (8) Klose, J. Humangenetik 1975, 26, 231-243. (9) Davis, M. T.; Beierle, J.; Bures, E. T.; McGinley, M. D.; Mort, J.; Robinson, J. H.; Spahr, C. S.; Yu, W.; Luethy, R.; Patterson, S. D. J. Chromatogr., B 2001, 752, 281-291. (10) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683-5690. (11) Naegele, E.; Vollmer, M.; Hoerth, P. J. Chromatogr., A 2003, 1009, 197205. (12) Liu, H., Lin, D.; Yates, J. R., III. BioTechniques Eur. Ed. 2002, 32, 898911. (13) Wu, C. C.; Yates, J. R., III. Nat. Biotechnol. 2003, 21, 262-267. 10.1021/ac040022u CCC: $27.50

© 2004 American Chemical Society Published on Web 08/06/2004

The orthogonal combination of HPLC techniques that separate samples according to different molecular properties (2DLC or multidimensional LC) has been outlined for proteins and peptide mixtures in a variety of reports.14,15 In general, peak capacity for a multidimensional separation is calculated by multiplying the peak capacity of the single-separation steps.16 By combining two different LC techniques with individual peak capacities between 50 and 100, it will be theoretically possible to achieve total capacities for the separation of 2500-10 000 peaks. Complex protein expression patterns have been analyzed either after the separation was conducted on the protein level or alternatively on the peptide level.14 The latter approach is more easily automated since protein digestion of the sample takes place at the beginning of the work flow. In addition since large and hydrophobic proteins tend to adsorb to surfaces of the separation devices and tubings, this is much less pronounced when working with peptides. In contrast conducting proteome separation exclusively at the peptide level increases the initial complexity of the starting sample dramatically by increasing the total amount of different components by 1-2 orders of magnitude. The separation of complex peptide mixtures has been achieved by the combination of several orthogonal techniques: (i) reversedphase chromatography (RP)-capillary electrophoresis;17 (ii) size exclusion chromatography (SEC)-RP;18 (iii) strong cation exchange chromatography (SCX)-RP.9,10 Most reports describe a separation procedure where the SCX step is performed “on-line” with the second separation step (RP). Such SCX-RP combinations have been applied successfully for the analysis of comprehensive protein expression profiles, e.g., from Saccharomyces cerevisiae and Plasmodium falciparum.19,20 Peptides from protein digests are eluted off the SCX column by sequential salt plugs with increasing ionic strength. After each salt step, peptides are concentrated on an enrichment column (or directly on the analytical RP column) and separation is performed on the analytical nanobore RP column with a gradient of increasing organic solvent concentration. Recently, it was demonstrated that the overall peak capacity of a 2DLC system can be increased significantly by using “offline” 2DLC.21 Here, the SCX chromatography is performed by applying a linear salt gradient with subsequent microfraction collection. This technique is especially promising for highly complex samples originating from total cell lysates or body fluids while the highly automated on-line technique provides sufficient resolution for affinity-selected or subcellular protein fractions. The following study demonstrates that by using a novel microfraction collection system in combination with nanoflow electrospray the overall peak capacity can be further significantly (14) Wang, H.; Hanash, S. J. Chromatogr., B 2003, 787, 11-18. (15) Issaq, H. J.; Conrads, T. P.; Janini, G., M.; Veenstra, T. D. Electrophoresis 2002, 23, 3048-3061. (16) Giddings, J. High Resolut. Chromatogr. 1987, 10, 319. (17) Moore, A. W.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3448-3455. (18) Opiteck, G. J.; Ramirez, S. M.; Jorgenson, J. W.; Moseley, M. A. Anal. Biochem. 1998, 258, 349. (19) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (20) Florens, L.; Washburn, M. P.; Raine, J. D.; Anthony, R. M.; Grainger, M.; Haynes, D.; Moch, J. K.; Muster, N.; Sacci, J. B.; Tabb, D. L.; Witney, A. A.; Wolters, D.; Wu, Y.; Gardner, M. J.; Holder, A. A.; Sinden, R. E.; Yates, J. R., III; Carucci, D. J. Nature 2002, 419, 520-523. (21) Peng, J.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. J. Proteome Res. 2003, 2, 43-50.

augmented by optimizing gradient length, analysis time, stationaryphase selection, and column size of the first and second dimensions. These considerations can also serve investigators to develop the optimal separation method for his specific proteome sample. EXPERIMENTAL SECTION Chromatographic and MS Equipment. All described instrumentation equipment was from Agilent Inc. (Palo Alto, CA). The HPLC system used for this study was composed of two complete LC systems, the 1100 Micro Fraction Collection System for the first dimension and the 1100 Nanoflow Proteomics Solution for the second separation dimension. The 1100 Micro Fraction Collection System consisted of a capillary pump with microvacuum degasser, thermostated microwell-plate autosampler, thermostated column compartment with a two-position/six-port microvalve, DAD with 80-nL flow cell, and a thermostated microfraction collector. For the second dimension, the 1100 Nanoflow Proteomics Solution is composed of a nanoflow pump with microvacuum degasser, a thermostated microwell-plate autosampler, a twoposition/six-port switching microvalve box with holder, and an additional quaternary pump for fast sample loading and washing, and a LC/MSD Trap XCT with the orthogonal nanospray source were used. As software packages ChemStation A10.02 for HPLC/MS, IonTrap Software 4.2 and Spectrum Mill MS Proteomics Workbench for MS data processing were installed. A Spectrum Mill protein score is calculated as the sum of the peptide scores of a identified protein. Peptide scores are the sum of bonus points for each assigned peak to allowed fragment ion type (the allowed fragment ion type and bonus is mass spectrometer-type dependent), for example +1, 0.75, 0.5, 0.25 for b/y, internal, b - H2O/y - H2O, a, immonium fragments, respectively. A penalty score is subtracted for each unassigned peak - (peak height/height of tallest peak). The typical work flow for an off-line 2D LC proteome analysis is summarized as follows: In the first LC separation dimension, tryptic peptides are loaded onto the SCX column. Peptides are then eluted with a linear salt gradient with subsequent collection of fractions in the microliter range with the microfraction collector. The well plates containing the fractions are then transferred to the second dimensionsthe nano LC/MS system. After reinjection, the peptides from each fraction are concentrated on a short RP enrichment column located between two ports of the six-port microvalve. This enrichment column is then switched into the nanoflow path; the concentrated peptide fraction is eluted, further separated by a gradient with increasing organic solvent on an analytical nanobore column, and sprayed directly into the MS. Chromatography Methods. Off-Line 2D LC. First-Dimension (SCX Chromatography). For the first dimension, buffer A contained 5% AcN + 0.03% formic acid and buffer B 5% AcN, 500 mM NaCl + 0.03% formic acid. Typical flow rates were 5 µL/min unless otherwise stated. Gradients were conducted as discussed in Results and Discussion. For the analyses, the following columns were used: BioSCX Series II (Agilent), 35 × 0.3 mm, 3.5-µm particles as the standard column (or other dimension as indicated). Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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Sample volume was 5 µL, fractions of 15 µL were collected in time-based collection mode (first fraction 0-5 min, second to 13th fraction each 3 min). The fraction collector was set to liquid contact control mode, cooling was provided at 4 °C. Detection of eluting peptides using a diode array detector was performed simultaneously at 210, 222, and 280 nm (bandwidth 4 nm). The reference wavelength was set to 450 nm (band with 50 nm). Second-Dimension (RP Chromatography). The nanoflow conditions were as follows: solvent A, water + 0.1% formic acid; solvent B ACN + 0.1% formic acid. The gradients were run according to the following protocol: 0 min 5% B, 5 min 5% B, 12 min 15% B, 72 min 55% B, 74 min 75% B, 75 min 75% B, 75.01 min 5% B; stop time 90 min; post time 10 min. Flow was adjusted to 300 nL/min. For enrichment and analysis, following columns were used: Zorbax 300SB C18 or C8 (Agilent), 5 × 0.3 mm, 5-µm particles (or other dimensions as indicated in Results and Discussion); Zorbax 300SB C18, 150 mm × 75 µm, 3.5-µm particles (Agilent). The microvalve of the enrichment column was switched after 5 min of analysis start in order to direct the flow from the nanoflow pump through the enrichment column and analytical column. The injection volume of recovered fractions was 25 µL after adding 10 µL of water. MS Conditions: LC /MSD Trap XCT. Ionization mode was in positive nanoelectrospray with the orthogonal source. Drying gas flow was at 5 L/min and drying gas temperature 300 °C. Vcap was typically 1800-2000 V and the skimmer 30 V. The capillary exit offset was 75 V and trap drive 85 V. Maximum accumulation time was 150 ms, smart target 125 t000. The MS scan range was set between a mass of 300 and 2200. MS/MS was performed automatically in ultrascan mode (50-2200 m/z range can be scanned with 26 000 m/z per second) with three or four parents. Active exclusion was executed after four spectra for 1 min. Sample Preparation and Chemicals. Cell disruption was performed according to published protocols22 with cooled resuspended lyophilized E. coli cells (in 50 mM AmBic, 8 M urea) in a bead beater (BioSpec Products, Bartlesville, OK) with 0.1-mm glass beads. Proteins were reduced with 1 mM DTT, 37 °C, alkylated with 10 mM iodoacetamide for 30 min at room temperature, followed by ultrafiltration for buffer exchange (50 mM AmBic). Tryptic digest was done with TPCK trypsin (Pierce, Rockford, IL) at 37 °C for 16 h. Digested peptides were lyophilized and washed in deionized water followed by a second lyophilization using a SpeedVac (Bachofer, Reutlingen, Germany). Finally, the sample was dissolved in buffer A prior to analysis. All chemicals and bioreagents used in this study were obtained from Sigma GmbH (Taufkirchen, Germany). RESULTS AND DISCUSSION SCX Gradient Comparison. On the first separation dimension (SCX), four gradients differing in length were compared in order to obtain optimum resolution of the highly complex proteome peptide sample for a 2D LC run. Concurrently, the aim was to keep total analysis time of the complete 2D LC run in an acceptable time range. The first series of experiments comprises a digest of 10 bovine proteins as model system, the second series a digested E. coli lysate. Both were separated on a recently (22) Vollmer, M.; Na¨gele, E.; Ho ¨rth, P. J. Biomol. Techniques 2003, 14, 128135.

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Table 1. Peptide and Protein Identification of a 10-Protein Mix (upper) and an E. coli Lysate (lower panel) Depending on SCX Gradient Length SCX gradient (min)

run

first part (0-100 mM NaCl)

second part (100-500 mM NaCl)

no. of SCX fractions

1 2 3 4

15 30 60 120

(A) 10-Protein Mix 3 7 6 13 12 25 24 49

1 2 3 4

15 30 60 120

(B) E. coli Lysate 3 7 6 13 12 25 24 49

no. of peptides identified

no. of proteins detected

30 22 22 23

10 8 8 9

486 769 942 1081

182 268 312 339

developed SCX column material (35 × 0.3 mm, 3.5-µm particles), especially designed for separation of complex peptide mixtures. Linear salt gradients (0-100 mM NaCl) of 15, 30, 60, and 120 min were programmed followed by a steep short ramp up to 500 mM NaCl (for 2.5, 5, 10, and 20 min, respectively). Microfractions of 15 µL (3 min) were deposited in cooled 96 conical-shaped well plates by the liquid contact control mode of the microfraction collector, which is an instrument feature that ensures that the capillary of the fraction collector is in contact only with the liquid surface of the deposited droplet in order to prevent crosscontamination. Conditions for reinjection onto the second dimension and the second dimension itself were kept constant for all four gradients. The number of identified proteins clearly demonstrate that for relatively simple mixtures such as the artificial 10- protein mix a short steep gradient clearly provides sufficient resolution and no further increase is achieved by a more shallow gradient (Table 1A). This certainly also holds true for low-complexity samples originating, for example, from a highly resolved 2D gel spot or from isolated protein complexes. For a complex protein expression analysis, however, longer gradients result in a significant increase of identified proteins as shown for the total E. coli lysate (Table 1B). However, this increase in numbers of identified peptides and proteins results in much longer total analysis time. The increase in detected peptides and corresponding proteins for the E. coli sample was especially pronounced between the 15-min and the 30-min gradient experiment. Applying a 30-min gradient resulted in the identification of 37% more peptides identified compared to the 15-min gradient. More shallow gradients (60- or 120-min gradient) increase the number of identified peptides by a further 19 and 13%. If total analysis is taken into account, the 30-min NaCl gradient seems to be a good compromise. In this case, analysis time from the first dimension and the serial separation of the obtained 13 fractions by subsequent Nanoflow LC/MS add up to a total analysis time for the off-line 2D LC run of 22 h. If further resolution is required, such as for the identification of lowabundant proteins, longer gradients (60 or 120 min) should be conducted alternatively. The tradeoff for longer gradients, however, is the significant extension of total analysis time. For the 60- and 120-min SCX gradients, the analysis time compared to the 30-min gradient 2D LC procedure is increased by a further

Table 2. Peptides and Proteins Identified Depending on SCX Column Dimension SCX dimens no. of no. of (length × i.d. flow rate fraction peptides proteins column in mm) (µL/min) size (µL) identified identified 1 2 3 4

5 × 0.3 35 × 0.3 50 × 0.75 150 × 0.75

5 5 20 20

15 15 60 60

167 1082 490 527

65 422 179 169

100 and 400%, resulting in a total of 44- and 88-h runs for a single 2D sample, respectively. The resolution of the SCX separation remains good even for the 120-min gradient. This is demonstrated by the fact that in all conducted gradient lengths single peptides elute in only one or two distinct SCX fractions. A comprehensive MS/MS fragmentation pattern for the peptides just above the score significance threshold indicates the quality and reliability of the overall analysis. To demonstrate reproducibility of the analyses, runs were performed in triplicate. For the 10-protein mix, analyses the average relative standard deviations (RSDs) of the amount of identified peptides and proteins were 3 and 6%, respectively. The different gradients applied for the E. coli sample resulted in an average RSD of 4.3% on the peptide level and 9.8% on the protein level. These values indicate good reproducibility for the overall identified number of components under the different conditions applied in the experiment. Detailed inspection of the individual peptides and proteins for the E. coli sample that were detected in each run demonstrated good reproducibility for the 60% peptides and proteins with the highest score, while for the 40% with the lower scores a higher degree of variation was observed. This was especially pronounced in short, steep gradients where the overall peak capacity was much lower than with long shallow gradients. SCX Column Dimensions. To achieve the maximum number of detected peptides and identified corresponding proteins from proteomic samples, the proper combination of column dimensions of the first and second LC separation dimensions is required.

Nanobore columns, e.g., with 0.075-mm internal diameter, in combination with nanoelectrospray provide by far the highest sensitivity and were therefore chosen for the RP separation. To avoid overloading from the first dimension on one hand and to provide enough material for the detection of lower abundant proteins on the other hand, four SCX column dimensions were chosen for evaluation (150 × 0.75, 50 × 0.75, 35 × 0.3, 5 × 0.3 mm). Gradients and flow rates were adjusted approximately according to column dimension in order to achieve good separation. Total analysis time was kept constant for all experiments. E. coli lysates were separated in the off-line mode as described earlier. Results demonstrate that usage of the 35 × 0.3 mm size SCX column in combination with the 0.075-mm-internal diameter nanobore RP column results clearly in the highest number of identified proteins and therefore seems most suitable in the abovedescribed setup for the two-dimensional separation of complex proteomic samples (Table 2). The small number of identified peptides with 5 × 0.3 mm is probably due to limited capacity of this short column size, the lower amount of identified peptides with the large diameter SCX columns (0.75 mm) could be explained by peak broadening with these columns in contrast. Trapping Column Materials and Dimensions. (a) C8 versus C18 Material. Trapping or enrichment columns are used to efficiently concentrate a large-volume sample either for onedimensional nano LC or for transfer of eluted fractions from the first separation dimension to the second. Enrichment on a trapping column allows in addition fast sample loading and washing off salt with high flow rates while flow is directed to waste. Sample desorption takes place after switching a valve with reversed flow and flow is directed with nanoflow rates into the analytical nanoflow path and finally into the MS instrument. This setup bears the advantage that relatively large columns in the first dimension can be combined with nanobore columns in the second separation dimension in an automated fashion. To test whether short-chain C8 or more hydrophobic C18 reversedphase material is more effective for the enrichment of peptides eluting from the first dimension in combination with a C18 analytical nanocolumn for the second-separation dimension, both materials were tested and compared.

Table 3. BSA Peptides Identified from a 10-Protein Mix Using C8 and C18 Material for the Enrichment Columns BSA peptides found with C8

score

(K) DDPHACYSTVFDKLK(H) (K) HLVDEPQNLIK(Q) (K) KQTALVELLK(H) (R) KVPQVSTPTLVEVSR(S) (R) LCVLHEKTPVSEK(V) (K) LGEYGFQNAULIVR(Y) (K) LKPDPNTLCDEFK(A) (K)LVNELTEFAK(T) (R) NECFLSHKDDSPDLPK(L) (K) QTALVELLK(H) (R) RPCFSALTPDETYVPK(A) (K) SLHTLFGDELCK(V) (K) YLYEIAR(R)

4.2 11.88 4.7 13.97 10.85 5.12 16.43 8.88 10.93 12.54 12.54 13.26 8.19 133.49

BSA peptides found with C18 (K) DDPHACYSTVFDKLK(H) (K) HLVDEPQNLIK(Q) (K) KQTALVELLK(H) (R) KVPQVSTPTLVEVSR(S) (R) LCVLHEKTPVSEK(V) (K) LKPDPNTLCDEFK(A) (K) LVNELTEFAK(T) (R) NECFLSHKDDSPDLPK(L) (K) QTALVELLK(H) (R) RPCFSALTPDETYVPK(A) (K) SLHTLFGDELCK(V) (K) YLYEIAR(R) (K) EACFAVEGPK(L) (R) FKDLGEEHFK(G) (R) ETYGDMADCCEKQEPER(N) (K) EYEATLEECCAK(D) (K) CCAADDKEACFAVEGPK(L)

score 5.34 15.84 4.67 14.76 8.98 16.25 13.58 12.12 7.64 14.59 12.77 9.16 12.37 11.94 8.25 6.2 5.77 180.23

MH+ matched (Da) 1796.816 1305.717 1142.715 1639.938 1540.805 1479.796 1577.752 1163.631 1902.854 1014.620 1881.906 1420.678 927.494 1108.498 1249.622 2119.789 1504.582 1930.751

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Figure 1. Base peak chromatogramm and RP gradient from a gradient with 1% ACN increase per minute for the 10-protein digest. Mobile phase, chromatographic conditions, and column dimensions are indicated in the Experimental Section. Table 4. Identified Number of Proteins and Peptides from a 2 DLC Run Using Different Dimensions of Trapping Columns and Injection Volumes 2 × 0.3 mm 5 × 0.3 mm 5 × 0.5 mm E. coli (µL) peptides proteins peptides proteins peptides proteins 0.1 1 10

31 146 166

12 51 65

44 157 165

15 56 69

76 152 151

Table 5. Identified BSA Peptides from a 10-Protein Mix Depending on Steepness of the Gradient of the RP Separation (Second Dimension)a gradient slope % B/min

total MS score

distinct peptides ((SD)

no. of spectra

% AA coverage ((SD)

2 1 0.5

141.27 265.05 174.68

14 ((0.7) 23 ((2.3) 15 ((1.3)

40 139 129

27 ((2) 44 ((5) 26 ((4)

27 63 67 a

Experiments were performed using a BSA digest (100 fmol) and an artificial 10-protein mix (100 fmol of each protein), respectively. Trapping columns with C18 material resulted in higher peptide scores and subsequently in a higher number of identified peptides than columns packed with C8 reversed-phase material. This holds true especially for the BSA digest (Table 3) and can also be observed with a 10-protein mix (data not shown). It indicates that C18 material is slightly superior to the C8 in trapping efficiency for peptide digests. Trapping Column Dimensions. Since enrichment columns are the interface between the first and second dimensions, it was critical to investigate the influence of column dimension in order to provide for a complete system and enough loading capacity to trap all material from the first dimension (without overloading the following nanocolumn) and to effectively concentrate the sample in a narrow band. Therefore, three different column sizes (2 × 0.3, 5 × 0.3, 5 × 0.5 mm) differing in length and diameter were investigated. E. coli sample was injected directly in different amounts (0.1, 1, 10 µL) onto the C18 trapping columns. While with the 2-mm column significantly lower amounts of peptides were identified for 0.1 µL of extract, all columns showed comparable results for a higher sample load. This indicates that the chosen standard trapping dimension of 5 × 0.3 mm properly fits in a 2D LC system with 35 × 0.3 mm on the SCX and 150 × 0.075 mm on the RP dimension, respectively (Table 4). Comparison of RP Gradients (the Second Dimension) on the Separation of Proteomic Samples. In multidimensional chromatography, which uses RP chromatography in the second dimension directly prior to MS analysis, it is most important that 5184 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

All experiments were performed in triplicate.

this separation step is effectively optimized because RP chromatography has a very high resolving power. A nanobore column of 75-µm internal ID was chosen since it allows optimum flow rates at ∼300 nL/min, which in conjunction with nanoelectrospray provides exquisite sensitivity on the MS detection site. Therefore, the optimum performance of this separation step will influence mostly the number of identified peptides and corresponding proteins. To investigate the nanobore reversed-phase chromatography, a mixture of 10 digested bovine proteins was used. The digest was separated with gradients delivering slopes ranging between 0.50 and 2% acetonitrile/min in a concentration range between 5 and 65% acetonitrile. To find out the relation between gradient steepness and number of identified peptides, acquired spectra, scores, and sequence coverage of BSA (as one component in the mixture) were compared for the applied gradients, respectively (Table 5). The maximum number of identified peptides and proteins were obtained with a slope of 1% acetonitrile/min. Results obtained from steeper gradients might suffer from a lack of resolution while more shallow gradients lead to more flat and broader peaks eluting into the ion trap MS instrument. The displayed base peak chromatogram demonstrates that the majority of the peptides elutes between 20 and 50 min between 15 and 45% acetonitrile (Figure 1). The optimized gradient therefore started with an initial steeper ramp up of acetonitrile to 15% followed by a shallow part between 15 and 55% acetonitrile in which the majority of peptides was eluting. Reproducibility of the results is demonstrated by performing all of the analyses in

triplicate (Table 5). In addition to the investigation of different gradients on the overall peak capacity, the influence of column length would be another parameter that could be examined and that would definitely complete the overall picture of the 2 D LC/ MS system. However, a 150-mm column was chosen as standard since it definitely provides a high plate number in combination with an acceptable back pressure at 300 nL/min. CONCLUSION The combination of strong cation exchange chromatography with nanobore RP chromatography in two-dimensional off-line HPLC for proteomic samples requires a careful design of gradients and column dimensions in order to achieve maximum resolution.

For high-complexity samples, increase in gradient length on the SCX dimension clearly increases the number of identified proteins. This however also increases significantly total analysis time. The selection of 35 × 0.3 mm SCX column containing BioSCX series II material in combination with 5 × 0.3 mm enrichment columns with C18 material and a 150 × 0.075 mm nanobore column with the same material proves to be an excellent choice to perform off-line 2D LC in the described system configuration.

Received for review February 6, 2004. Accepted June 17, 2004. AC040022U

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