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Letters to Analytical Chemistry Quantitative Improvements in Peptide Recovery at Elevated Chromatographic Temperatures from Microcapillary Liquid Chromatography-Mass Spectrometry Analyses of Brain Using Selected Reaction Monitoring Santiago E. Farias,† Kelli G. Kline,‡ Jacek Klepacki,† and Christine C. Wu*,† Department of Pharmacology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, 80045, and Genetics and Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706 Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using selected reaction monitoring (SRM) mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in mouse whole brain matrix are highlighted, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in microcapillary liquid chromatography-mass spectrometry (µLC-MS) based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification. One of the major limitations for quantitative proteomic analyses of complex biological samples is low analytical throughput. Complex samples (such as mammalian tissues) are typically fractionated to enrich for low abundance proteins.1-3 However, * Corresponding author. Christine C. Wu, Ph.D. Assistant Professor of Pharmacology, University of Colorado School of Medicine, Department of Pharmacology, Mail Stop 8303 RC1-South Tower, L18-6117, 12801 East 17th Avenue, P.O. Box 6511, Aurora, CO 80045. Phone: 303-724-3351. Fax: 303-7243663. E-mail:
[email protected]. † University of Colorado School of Medicine. ‡ University of Wisconsin. (1) Fang Y. P., Robinson D. P., Foster L. J. Proteome Res. DOI: 10.1021/ pr901063t. Published Online: January 16, 2010. 10.1021/ac100359p 2010 American Chemical Society Published on Web 04/07/2010
fractionation increases the total number of samples to be analyzed, requires larger amounts of input tissue, and may lead to errors associated with reproducibility of fractionation and biased biochemical losses. Optimal throughput is achieved by bypassing the fractionation step. However, quantitative measurements in complex unfractionated tissue samples necessarily require improvements in analyte separation, recovery, and detection. Many current proteomic platforms are microcapillary liquid chromatographytandem mass spectrometry (µLC-MS/MS) based, and improvements in instrument performance, sensitivity, and analytical speed are continuously being realized.4 Likewise, improvements in µLC separations and chromatographic recovery can also contribute significantly to the throughput of analyses by enhancing the “apparent” sensitivity of the mass analysis. It is well recognized that conducting LC at elevated temperatures (typically 30-80 °C) results in beneficial effects, such as improvements in peak shape, selectivity, and resolution.5,6 Furthermore, elevated temperatures are sometimes required for the successful elution of very hydrophobic proteins.7 Previously, we demonstrated that elevated chromatographic temperature enhances the recovery and therefore the identification of hydrophobic peptides from enriched plasma membrane samples using undirected µLC-MS/MS analyses.8 This study utilized peptide identification from tandem mass spectra acquired from shotgun analyses as the metric for improvements in total peptide recovery. However, quantitative measurements for each peptide analyte (2) Yates, J. R., 3rd; Gilchrist, A.; Howell, K. E.; Bergeron, J. J. Nat. Rev. Mol. Cell. Biol. 2005, 6 (9), 702–714. (3) Wu, C. C.; MacCoss, M. J.; Mardones, G.; Finnigan, C.; Mogelsvang, S.; Yates, J. R., 3rd; Howell, K. E. Mol. Biol. Cell 2004, 15 (6), 2907–2919. (4) Schmidt, A.; Claassen, M.; Aebersold, R. Curr. Opin. Chem. Biol. 2009, 13 (5-6), 510–517. (5) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development, 2nd ed.; Wiley & Sons: New York, 1997. (6) Dolan, J. W. J. Chromatogr., A 2002, 965 (1-2), 195–205. (7) Martosella, J.; Zolotarjova, N.; Liu, H.; Moyer, S. C.; Perkins, P. D.; Boyes, B. E. J. Proteome Res. 2006, 5 (6), 1301–1312. (8) Speers, A. E.; Blackler, A. R.; Wu, C. C. Anal. Chem. 2007, 79 (12), 4613– 4620.
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Table 1. Selected Synthetic Peptidesa
a
mouse protein
GRAVY
peptide sequence
m/z
transition 1
transition 2
Ce63-9 Ppm1-b1 Homer-20 Paqr7-1 P2RX4-2 Gria-1 Homer-26 Homer-6 Homer-2P FABP3-2 Homer-21 Rdh11-2 DAT-2 Gria2 Gnmt-M3 Rdh11-4 FABP3-3 DAT-4
-0.82 -0.69 -0.57 -0.46 -0.39 0.07 0.27 0.42 0.50 0.64 0.69 1.07 1.32 1.36 1.40 1.41 1.48 1.57
LLEALQEEQK YGLSSMQGWR MGEQPIFTTR PEPVFTVDR AFLFEYDTPR AWNGMVGELVYGR SFLEVLDGK VIINSTITPNMTFTK IALTQSAANVK SLGVGFATR QAVTVSYFYDVTR LANILFTK IDFLLSVIGFAVDLANVWR ADVAVAPLTITLVR AWLLGLLR WLWQLFFVFIK LILTLTHGSVVSTR PYVVLTALLLR
600.83 592.78 590.30 530.28 629.81 726.36 504.27 840.46 558.32 454.25 774.89 460.28 1074.60 719.94 471.30 763.93 748.00 629.40
600.83/974.48 592.78/851.38 590.30/734.42 530.28/833.45 629.81/927.42 726.36/793.42 504.27/773.44 840.46/939.46 558.32/818.43 454.25/707.38 774.89/1050.49 460.28/735.44 1074.60/1247.65 719.94/912.59 471.30/684.48 763.93/913.55 748.00/705.38 629.40/898.61
600.83/845.44 592.78/764.40 590.30/524.28 530.28/637.33 629.81/780.35 726.36/892.49 504.27/40/660.36 840.46/838.41 558.32/589.33 454.25/551.29 774.89/1250.61 460.28/806.48 1074.60/1360.74 719.94/983.62 471.30/571.39 763.93/1227.69 748.00/842.44 629.40/799.54
Summary of parent mouse protein, GRAVY score, peptide sequence, precursor m/z, and SRM transitions monitored.
were not assessed, and the magnitude of peptide recovery was not determined. In this study, we used targeted mass spectrometry to quantify the detection of 18 synthetic peptides of a wide range of hydrophobicities when added to both a simple peptide matrix (derived from 5 proteins) and a complex peptide matrix (derived from mouse brain) during standard reversed-phase chromatography conducted at 25, 40, and 60 °C. Our results demonstrate that increased chromatographic temperature results in significant improvements in peptide recovery for all 18 peptides (hydrophilic and hydrophobic) monitored in both the simple and complex peptide matrix. On average, peptide recovery increased by 5-fold and 10-fold in the low and high complexity matrices, respectively, at elevated chromatographic temperatures. Specifically, the improvements demonstrated in the whole brain peptide matrix have important implications regarding the utility of this approach for complex biological applications. Because improvements in peptide recovery from chromatographic separations translate directly to improvements in mass spectrometric analyses for µLC-MS/MS based proteomic applications, the simple incorporation of elevated chromatographic temperatures will provide significant improvements in peptide quantification, as well as detection and identification, in complex mammalian tissues. METHODS Reagents. All solvents were purchased from Fisher Scientific (Pittsburgh, PA), and all chemicals were purchased from SigmaAldrich (St. Louis, MO) unless otherwise indicated. Synthetic peptides with crude purity (
