Application Note. Comparison of HPLC-Chip/MS with conventional nanoflow LC/MS for proteomic analyses

Transcription

1 Comparison of HPLC-Chip/MS with conventional nanoflow LC/MS for proteomic analyses Application Note Martin Vollmer Christine Miller Georges L. Gauthier Abstract Nanoflow LC/MS is commonly used for protein identification and biomarker discovery, but setting up and maintaining the intricate LC plumbing can be a challenge. The Agilent HPLC-Chip represents breakthrough technology that integrates nanoflow HPLC columns, associated connections, and a nanoelectrospray emitter on a single, small, reusable microfluidic chip. The HPLC-Chip and a companion HPLC- Chip/MS interface afford much better ease-of-use than traditional systems. This Application Note demonstrates performance of the HPLC-Chip/MS versus a state-of-the-art conventional nanoflow LC/MS. A direct comparison shows that the HPLC-Chip/MS system produces better chromatography and sensitivity, leading to more identified peptides and proteins.

2 Introduction Nanoflow LC/MS provides sensitive, highly specific analyses of complex proteomic samples. It is often the method of choice for this type of sample despite challenges associated with the number of plumbing connections and the need to keep the system completely free of leaks, blockages, and excessive dead volumes. A typical setup is comprised of relatively complex plumbing that includes an enrichment column to remove salts and concentrate the sample, an analytical column, a valve to switch the flows between the columns, a nanoelectrospray emitter, and the capillaries and fittings to connect them. Now most of these components have been integrated on a single microfluidic chip, the HPLC-Chip. A companion HPLC-Chip/MS interface includes the valve for flow switching, as well as everything else needed to couple the HPLC-Chip to the Agilent 1100 Series HPLC system, and the LC/MSD Trap. Smaller than a credit card, the Agilent HPLC-Chip consists of a polyimide-based chip material with microfluidic channels, appropriate HPLC column packings, an integral nanoelectrospray emitter, and electrical contacts for electrospray. The chip design significantly reduces the number of capillaries and fittings needed for nanoflow LC/MS. It is reusable, easier to set up and use, and is more reliable. This Application Note will demonstrate performance of the HPLC- Chip/MS versus conventional nanoflow LC/MS for proteome analysis of yeast (Saccharomyces cerevisiae). This work will illustrate that the chip format delivers reduced sample losses and lower delay volumes, thus producing uncompromised chromatographic performance and more identified peptides and proteins. In sum, the HPLC-Chip/MS system provides better results than a state-of-theart conventional system, with much better ease-of-use and reliability. Experimental Figure 1 shows the components and flows for a typical nanoflow LC/MS system. Such a system requires flow switching so that protein digests can be loaded using higher flow rates onto the enrichment column, flushed onto the analytical column, and then analyzed by nanoflow LC/MS/MS. The HPLC-Chip, in combination Loading pump Injector with the HPLC-Chip/MS interface, replaces the columns, intricate plumbing connections, and flowswitching valve needed for this setup. Fabrication and layout of the HPLC- Chip/MS The HPLC-Chip/MS is fabricated from polyimide film, a material that is resistant to most solvents, tolerates a wide ph range, and is compatible with the analysis of proteins and peptides. The fabrication process uses UV laser ablation in combination with vacuum lamination of the polyimide film to create a multilayer microfluidic device. 1 Open micro-channels are packed with reversed-phase column materials to create HPLC columns. Metals are applied by thin film deposition onto the polymer film surfaces to produce the electrical contacts for electrospray. Enrichment column C18 Nano column Waste Nanoflow LC pump Figure 1 Flow diagram for a conventional nanoflow LC/MS system with sample enrichment. Online MS/MS 2

3 Figure 2 shows a schematic of the HPLC-Chip. The chip format is very flexible and allows for various combinations of sample cleanup and one-dimensional or two-dimensional nanoflow HPLC analyses. For the experiments described here, the following components were integrated onto the HPLC-Chip: A 40-nL enrichment column packed with ZORBAX 300SB- C18, 5 µm particle size An analytical column packed with ZORBAX 300SB-C18, 5 µm particle size All connections between the two columns and between the analytical column and the nanoelectrospray tip The nanoelectrospray emitter (10 µm ID) Interface with the ion trap mass spectrometer The HPLC-Chip inserts into the HPLC-Chip/MS interface, shown in figure 3. This interface connects the Agilent 1100 Series nanoflow LC system and the LC/MSD Trap. It consists of the HPLC-Chip loading and ejection mechanism, a microvalve for flow switching, and nano-lc connections to the Agilent nano-lc pump and micro well-plate autosampler with loading pump. The HPLC-Chip/MS interfaces mounts to the electrospray source, which includes a miniature CCD camera for spray visualization. The HPLC-Chip/MS interface is a standard module within the Agilent 1100 Series LC system and is fully controlled by the Agilent ChemStation software. The HPLC-Chip/MS can also be interfaced to the Agilent LC/MSD TOF and has been applied to biomarker studies. 2 Waste Sample in Figure 2 Diagram of the HPLC-Chip. Sample Nano-LC pump enrichment column Figure 3 HPLC-Chip/MS Ion Trap system. Fluid connections from nanolc Multi-port rotary valve Rotor Stator The HPLC-Chip can be replaced in seconds. When an HPLC-Chip is loaded, leak-tight fluid connections are automatically established as the chip is sandwiched between the rotor and stator of the built-in multi-port microvalve. The rotor and stator dock onto LC column Top view: Analysis mode (flow path in red) the chip and establish a flow path from the nano-lc to the ports on the chip surface (figure 4). Fast movement of the rotor ensures reliable switching between sample loading and sample analysis positions on the HPLC-Chip. The loading mechanism in the HPLC- HPLC- Chip/MS interface HPLC-Chip Side view (fluid path in red) Figure 4 The microvalve in the HPLC-Chip/MS interface docks to an HPLC-Chip. LC/MSD Trap Nanospray tip HPLC-Chip (not to scale) 3

4 Chip interface precisely and automatically positions the nanoelectrospray emitter orthogonal to the MS inlet for maximum sensitivity and robustness. Comparison of conventional LC/MS with HPLC-Chip/MS Conventional nanoflow LC/MS was directly compared to HPLC- Chip/MS for the analysis of the yeast proteome. The proteins from the cell extract were first separated by SDS-PAGE. Fifteen micrograms of protein was loaded in each lane of the gel. A single gel band was excised. Resulting peptides were isolated using the Agilent Protein In-Gel Tryptic Digestion Kit (p/n ), as described in the corresponding protocol. The sample was then subjected to analysis by nano- LC/MS and HPLC-Chip/MS. To prevent sample degradation, aliquots of the sample were stored at 18 ºC and were freshly thawed for each set of experiments. Sensitivity of the instrument was monitored by injecting 10 fmol of BSA digest prior to and after every set of experiments, and comparable sensitivity was confirmed by identification of approximately the same number of peptides each time. Chromatographic conditions, gradients, and column lengths and materials were identical for the chip and the 43-mm nanocolumn in the conventional nano-lc system. In addition, the chip was compared to a standard nanocolumn of 150-mm length. The gradient used for the latter experiment was adapted to the length of the column. Average # of identified peptides or proteins Comparison of HPLC-Chip/MS and conventional analysis with nanocolumns Standard chip, 43 mm, 5-μm particle size Peptides All analyses were performed on the same instrument using either an orthogonal nanospray ion source or an HPLC-Chip/MS interface. All analyses used the same Agilent 1100 Series HPLC samplers and pumps, and the same Agilent LC/MSD Trap XCT ion trap mass spectrometer. Data reduction was accomplished with the Agilent Spectrum Mill MS Proteomics Workbench. Identical procedures and criteria were chosen in all experiments for MS and MS/MS data processing, protein database searches, and results validation. Every analysis was performed in triplicate. 79 Comparable column, 43 mm, 5-μm particle size Standard column, 150 mm, 3.5-μm particle size Proteins Figure 5 Average number of identified peptides and proteins from yeast gel band using the HPLC-Chip/MS (43 mm) versus conventional LC/MS with nanocolumns (43 mm and 150 mm). Results and Discussion More identified proteins with the HPLC-Chip/MS The results of the analyses of the gel band are shown in figure 5. The average numbers of identified peptides and proteins are presented for the triplicate runs. The results clearly demonstrate that the number of identified peptides and the number of detected proteins were significantly greater with the HPLC-Chip/MS system than with the conventional nanoflow LC/MS system. This was the case not only when the HPLC- Chip and nanocolumn of similar dimensions were compared (43-mm length), but also when the HPLC-Chip was tested against a 7 4

5 standard nanocolumn of 150-mm length. The superior results with the HPLC-Chip are attributable to better chromatography and improved sensitivity. Comparison of base peak chromatograms taken under the three different conditions demonstrate increased resolution and superior peak shape for the separation performed with the HPLC-Chip. (figure 6.) Optimized sample pathway A number of factors contributed to the improved analysis with the HPLC-Chip/MS. First, since integration of components on the Intens. x x x Standard HPLC-Chip, 43 mm, 5.0-μm particles Comparable nanocolumn, 43 mm, 5.0-μm particles Standard nanocolumn, 150 mm, 3.5-μm particles Time [min] Figure 6 Comparison of base peak chromatograms from analysis of the same sample using the HPLC- Chip/MS (43 mm) and conventional LC/MS with nanocolumns (43 mm and 150 mm). Chromatographic conditions Columns for the conventional nanoflow HPLC/MS system: Enrichment column: ZORBAX 300SB-C18, 300 µm x 5 mm, 5 µm (p/n ) Analytical columns: ZORBAX 300SB-C18, 75 µm x 150 mm, 3.5 µm (p/n ) or ZORBAX 300SB-C18, 75 µm x 43 mm, 5 µm HPLC-Chip/MS (p/n G ): Enrichment column: ZORBAX 300SB-C18, 40 nl, 5 µm Analytical column: ZORBAX 300SB-C18, 75 µm x 43 mm, 5 µm Loading flow rate: 4 µl/min Loading mobile phase: 2 % acetonitrile, 0.1 % formic acid Loading time (column switch): 4 min Injection volume: 2 µl Valve positions: 0 min enrichment, 4 min analysis, stop time enrichment Flow rate: Mobile phase: 300 nl/min A = 0.1 % formic acid in water B = 0.1 % formic acid in 99.9 % acetonitrile Gradient: Time % B 45-mm column 150-mm or chip column Stop time Needle flush solvent: 50 % methanol % formic acid in water Bottom-sensing: On, 1 mm offset for the plastic micro-insert vials MS conditions Ionization mode: Positive nanospray with Agilent orthogonal source (G1982B) or HPLC-Chip/MS interface (G4240A) 4 L/min Drying gas flow: Drying gas temperature: 320 C Capillary voltage: 1900 V for conventional nanoflow HPLC, 1800 V with HPLC-Chip Skimmer 1: 30 V Capillary exit: 100 V Trap Drive: 85 Averages: 2 ICC: On Maximum accumulation time: 150 ms Smart Target: 125,000 MS scan range: Automatic MS/MS: Peptide scan mode (standard-enhanced for MS and ultra-scan for MS/MS) Number of precursors: 3 Averages: 2 Fragmentation amplitude: 1.3 V SmartFrag: On, % Active Exclusion: Prefer +2: MS/MS Scan Range: ICC target: 125,000 On, 2 spectra, 1 min On Spectrum Mill MS Proteomics Workbench settings MS/MS search settings: Database: SwissProt, yeast subset database Search mode: Identity Digest: trypsin Maximum # missed cleavages: 1 N-terminus: hydrogen C-terminus: free acid Data validation: Autovalidation only, with the following scores: Protein: 13 Peptide: 9 SPI % : 70 5

6 Distinct Mean Distinct Summed Peptide Group Spectra Peptides MS/MS Search % AA Spectral Database (#) (#) (#) Score Coverage Intensity Accession # Protein Name E+08 P00924 Enolase 1 (EC ) (2-phosph0glycerate dehydr E+07 P00560 Phosphoglycerate kinase (EC ) E+07 P00890 Citrate synthase, mitochondrial precursor (EC E+07 P06106 MET17 protein [Includes: O-acetylhomoserine sulfhy E+07 P02994 Elongation factor 1-alpha (EF-1-alpha) E+07 P10592 Heat shock protein SSA E+07 P07256 Ubiquinol-cytochrome C reductase complex core prot E+07 P00549 Pyruvate kinase 1 (EC ) E+07 P39954 Adenosylhomocysteinase (EC ) (S-adenosyl-L E+06 P41939 Isocitrate dehydrogenase [NADP] cytoplasmic (EC E+07 P16547 Mitochondrial outer membrane 45 kda protein E+07 P19262 Dihydrolipoamide succinyltransferase component of E+07 P46672 GU4 nucleic-binding protein 1 (G4p1 protein) (P42) E+06 P08679 Citrate synthase, peroxisomal (EC ) E+06 P48570 Homocitrate synthase, cytosolic isozyme (EC E+07 P36008 Elongation factor 1-gamma 2 (EF-1 gamma 2) E+06 P00830 ATP synthase beta chain, mitochondrial precursor ( E+06 P39683 Probable nicotinate phosphoribosyttransferase (EC E+06 P phosphogluconate dehydrogenase, decarboxylating E+07 P38999 Saccharopine dehydrogenase [NADP+, L-glutamate for E+06 P06169 Pyruvate decarboxylase isozyme 1 (EC ) E+06 P methyltetrahydropteroyltriglutamate--homocystein E+07 P11484 Heat shock protein SSB1 (Cold-inducible protein YG E+06 P40054 D-3-phosphoglycerate dehydrogenase 1 (EC ) Totals: Figure 7 Twenty-four proteins identified from triplicate analyses of yeast gel band on an HPLC-Chip with a 43-mm column path. Distinct Mean Distinct Summed Peptide Group Spectra Peptides MS/MS Search % AA Spectral Database (#) (#) (#) Score Coverage Intensity Accession # Protein Name E+07 P00560 Phosphoglycerate kinase (EC ) E+07 P00925 Enolase 2 (EC ) (2-phosphoglycerate dehydr E+07 P06106 MET17 protein [Includes: O-acetylhomoserine sulfhy E+07 P02994 Elongation factor 1-alpha (EF-1-alpha) E+06 P00890 Citrate synthase, mitochondrial precursor (EC E+06 P16547 Mitochondrial outer membrane 45 kda protein. Figure 8 Six proteins identified from triplicate analyses of the same yeast gel band on a conventional LC/MS system with a 43-mm nanocolumn. HPLC-Chip eliminated most plumbing connections, dead volumes were reduced. Second, sample adsorption was minimized by the use of biocompatible polyimide and the elimination of troublesome connectors susceptible to sample adsorption. Third, since the electrospray emitter was integrated into the HPLC-Chip, postcolumn peak dispersion was negligible. Overall, the optimized design of the sample pathway minimized sample loss and reduced dead volume. These enhancements significantly increased the number of identified peptides and proteins with the HPLC-Chip format. Better sequence coverage with the HPLC-Chip/MS For proteins detected by both the HPLC-Chip/MS and conventional nanoflow LC/MS, sequence coverage was generally greater with the HPLC-Chip/MS. Better sequence coverage provided a higher level of confidence in the protein identifications. The proteins detected by HPLC-Chip/MS and the nanocolumn of comparable dimension are shown in figures 7 and 8, respectively. 6

7 Excellent reproducibility An additional benefit observed with HPLC-Chip/MS was a high degree of reproducibility between repetitive analyses. Figure 9 demonstrates this for the proteins that were identified from database search of three sequential runs of the same sample. Fifteen out of twenty-one proteins were found in all the analyses and only two proteins were identified from a single analysis. On the peptide level this was even more striking, as exemplified by the protein citrate synthase (figure 10). Among the ten identified peptides for this protein, nine were found in all analyses, while only one was detected in a single analysis. # of identified proteins Found in 3/3 runs Found in 2/3 runs Found in 1/3 runs Figure 9 Reproducibility of identified proteins from triplicate runs with HPLC-Chip/MS. Distinct Mean Distinct Summed Peptide Group Spectra Peptides MS/MS Search % AA Spectral Protein MW Database (#) (#) (#) Score Coverage Intensity (Da) Accession # Protein Name e P00890 Citrate synthase, mitrochondrial precurseor (EC 4.1) (#) C C C Intensity Intensity Intensity e e e e e e e e e e e e e e e e e e e e e e e e e e e e+007 Figure 10 Reproducibility of identified peptides for citrate synthase from triplicate runs with HPLC-Chip/MS. Conclusion The integration of sample enrichment, separation, and nanoelectrospray tip on the HPLC-Chip enabled much greater ease-of-use, better reliability, and faster setup than traditional nanoflow LC/MS systems. The integrated system minimized sample path length and peak dispersion, affording greater sensitivity. A direct comparison of conventional nanoflow LC/MS with the HPLC-Chip/MS on the same instrument showed that the HPLC-Chip/MS system produced more protein identifications with better sequence coverage and with a high degree of reproducibility from run to run. 7

8 References 1. Yin H., Killeen K., Brennen R., Sobek D., Werlich M., and van de Goor T., Microfluidic Chip for Peptide Analysis with an Integrated HPLC Column, Sample Enrichment Column, and Nanoelectrospray Tip, Anal. Chem., 77 (2), , Fortier, M-H, Bonneil, E., Goodley, P., and Thibault, P., Integrated Microfluidic Device for Mass Spectrometry-Based Proteomics and Its Application to Biomarker Discovery Programs, Anal. Chem., 77(6), , Martin Vollmer is an R&D Scientist at Agilent Technologies, Waldbronn, Germany. Christine Miller is a Senior Application Chemist at Agilent Technologies, Santa Clara, California, USA. Georges L. Gauthier is the Product Manager for the HPLC-Chip Technology program at Agilent Technologies, Waldbronn, Germany. Copyright 2005 Agilent Technologies All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws. Published August 1, 2005 Publication Number EN