Increased Throughput and Reduced Carryover of Mass Spectrometry-Based Proteomics Using a High-Efficiency Nonsplit Nanoflow Parallel Dual-Column Capillary HPLC System Hong Wang* and Samir M. Hanash Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Received December 21, 2007
We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 µm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet g 0.9, and peptides matched g 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events. Keywords: Parallel-Column capillary HPLC • nonsplit • nanoflow • ESI LTQ-FTICR MS • Plasma Proteomics • Biomarker
Introduction Proteomics is a fast moving field with innovations aimed in part at increasing sensitivity and throughput. High-efficiency, high-resolution, multidimensional procedures used in an integrated and automated fashion are needed to achieve sufficient depth of analysis of complex mixtures such as biological fluids for discovery studies. The protein constituents of plasma extend across 10-12 orders of magnitude in concentration. This presents a daunting hurdle because no single analytical technique or instrument has a dynamic range that can accommodate such wide concentrations. Multidimensional liquid chromatography is a useful strategy to increase coverage in proteomics. One approach termed MudPIT relies on two* To whom correspondence should be addressed. Hong Wang, Ph.D., Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109. Phone, 206-667-5716; fax, 206-667-2537; e-mail,
[email protected]. 10.1021/pr700876g CCC: $40.75
2008 American Chemical Society
dimensional separations of protein digests followed by on-line MS/MSMS analysis.1–3 However, analysis of random peptides from complex digests may not be sensitive to protein posttranslational modifications and other types of protein processing and may therefore miss protein isoforms associated with disease states.4–6 To address these issues, we have developed an orthogonal intact protein separation-based quantitative analysis system (IPAS) for biomarker discovery,5 following the mathematical model introduced by Giddings.7 We subsequently incorporated stable isotopic labeling into IPAS for quantitative plasma proteomic analyses.8 We have utilized this automated orthogonal 2D-HPLC system (anion-exchange chromatography as the first dimension, and reversed-phase chromatography as the second dimension) to fractionate plasma following depletion of abundant proteins and isotope-labeling of paired plasma samples (control versus disease) at an intact-protein level. The resolved protein fractions from the 2D-HPLC separations are Journal of Proteome Research 2008, 7, 2743–2755 2743 Published on Web 05/31/2008
research articles subjected to in-solution trypsin digestion and to 1D LC-MS/ MSMS analysis. Although the high-abundance proteins (i.e., albumin, IgG, IgA, transferrin, haptoglobin, and R-1-antitrypsin) which consist roughly of 85% of the total protein mass in plasma are removed by immunodepletion chromatography, the dynamic range of abundance of the remaining plasma proteins in 2D-HPLC fractions still spans some 6 orders of magnitude.9 Excessive loading of high-abundance proteins may interfere with the analysis of lower-abundance proteins in the datadependent acquisition (DDA) mode for MSMS. To address this issue, we use longer reversed-phase capillary columns (25 cm packing length) to increase resolution and peak capacity. Another issue is sample carryover from previous LC-MS runs, which complicates the analysis and reduces the efficiency and lifetime of capillary columns. Carryover is due to partial elution of high-abundance peptides and large peptides that bind tightly to the capillary column, have limited solubility in the usual water/acetonitrile/0.1% formic acid mobile phase, and have a propensity to aggregate. Thus, an extensive wash run has to precede an LC-MS run, which reduces throughput and the MS duty cycle. The need to increase LC-MS analysis throughput has driven the development of multicolumn parallel HPLC systems for either isocratic or gradient methods. An interface to multiplex as many as eight streams from eight parallel conventional HPLC columns into the regular ESI source of a single mass spectrometer has been commercialized and is widely used in compound screening for early drug discovery.10 A comprehensive on-line 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight
