1. Results Protein Removal is shown in the silver stained SDS-PAGE gel in Figure 3 below: F IGURE 3: A 10% SDS-PAGE gel after silver staining showing the amount of intact protein remaining after enriching plasma samples. These results show that the two best ways to remove intact protein are precipitation with two volumes of acetonitrile (lane 6) and filtration (lane 7). These two methods were taken forward to assess peptide retention with mTRAQ™ labelled QconCAT™ peptide standards. Extracted ion chromatograms for the 113 (Δ 0 ) and 117 (Δ 4 ) mTRAQ™ reporter ions were used to determine peptide losses, as shown in Figure 4. F IGURE 4: An MS/MS spectrum showing the MRM responses for each transition used to identify each peptide. From this, differences in the mTRAQ™ reporter ion intensities can be clearly seen (insert center). By assessing the peak area for extracted ion chromatograms for each reporter ion (insert, right), peptide losses can be assessed. Peptide Loss data for all peptides tested is presented in the Table 1. To calculate this, the heavy peak (peptide spiked into plasma before protein depletion, red in Figure 4) and the light peak (peptide spiked in after depletion, black in Figure 4) can be compared. Relative peak areas show the recovery of this peptide following protein removal. T ABLE 1: The results of peptide retention testing, showing the average recovery and variance for each target peptide when plasma is enriched by filtration or precipitation with two volumes of acetonitrile. The Use of Mass Spectrometry Combined with mTRAQ™-Labelled Surrogate Standards to Compare Enrichment Strategies for Target Peptides in Plasma David N. Potier 1, John R. Griffiths 2, Richard D. Unwin 3, Ralf Hoffman 4, Anthony D. Whetton 1 1: Stem Cell and Leukaemia Proteomics Laboratory (SCALPL), University of Manchester, UK, 2: Paterson Institute of Cancer Research, University of Manchester, UK, 3: Centre for Advanced Diagnostics and Experimental Therapeutics (CADET), Manchester,4: Philips Research, Eindhoven, The Netherlands. Method Plasma was obtained from healthy volunteers and pooled. The plasma was then centrifuged at 13,000 rpm for 5 minutes at 4 o C. The supernatant was removed, diluted four-fold with PBS and spin filtered (0.22 µm cut-off) at 4,000 rpm for 30 seconds at 4 o C. The resulting filtrates were pooled and aliquoted out into 25 µl samples. Each sample had a Δ 4 mTRAQ™ labelled series of peptide standards derived from a QconCAT™ protein added to it (1 pM) and enriched for peptides in one of the following ways. Peptide Enrichment Precipitation: Samples had either one or two volumes of solvent added to them and were vortexed for 10 seconds. The resulting solution was then centrifuged at 10,000 rpm for 30 seconds. The supernatant was removed and saved for analysis. Filtration: Samples were diluted with PBS to 1 ml and placed in the filter (5 kDa M W cut-off) and centrifuged at 3,200 rpm at 10 o C for 30 minutes. C 8 Extraction: Samples were prepared by adding C 8 coated magnetic beads, agitating, removing the beads and treating with 0.1% (w/v) TFA. These samples were then dried to completion and re-suspended in PBS. All samples were analysed by SDS-PAGE and silver staining to assess the amount of protein remaining in the sample after enrichment as shown in Figure 1. F IGURE 1: The workflow to monitor protein removal by each peptide enrichment method assayed in this study. The methods with the least amount of protein present after enrichment were re-prepared, and peptide recovery was analysed as follows. F IGURE 2: The workflow to monitor peptide retention by the methods most efficient at protein removal. References 1: Kay R, et al. Rapid Commun Mass Spectrom. 2008 Oct;22(20):3255-60. 2: Chertov O, et. al. Expert Rev Proteomics. 2005 Jan;2(1):139-45. 3: Alpert et. al., ABRF Poster, 2003 4: Tirumalai RS, et. al. Mol Cell Proteomics. 2003 Oct;2(10):1096-103. 5: Villanueva J, et. al. Mol Cell Proteomics. 2008 Mar;7(3):509-18. Acknowledgements We thank BBSRC & Philips for funding this work. We also thank Dr. Duncan Smith, Dr. Michael Walker & Mrs. Yvonne Connolly for their input in many helpful discussions. We also thank Chemicell for the gift of C 8 magnetic beads. Conclusion The best way to remove intact protein from a plasma sample was either precipitation with two volumes of acetonitrile or by filtration. The method offering the most reproducible peptide recovery results was precipitation with two volumes of acetonitrile. The optimal method to both remove protein and retain peptides was precipitation with two volumes of acetonitrile. 1 = M W Markers 2 = Untreated Plasma 3 = 1 vol. Acetone 4 = 2 vol. Acetone 5 = 1 vol. Acetonitrile 6 = 2 vol. Acetonitrile 7 = Filtration (5kDa cut-off) 8 = C 8 Extraction 1 2 3 4 5 6 7 8 Introduction Monitoring and quantifying lower abundance proteins and peptides in human plasma has become a goal of many research groups as this is expected to provide an accurate picture of the body’s response to a disease or treatment. However, the dynamic range of protein concentrations in plasma spans over 10 orders of magnitude, with 22 proteins making up over 99% of the total plasma protein content 1. Therefore, it is desirable to remove these high abundance intact proteins in order to probe deeper into the plasma peptidome in order to search for potential biomarkers or indicators of response to treatment. Several methods have been published in recent years describing how to do this 1-5. In this study we undertake a comparison of several approaches to peptide enrichment from complex mixtures including proteins. This study involves a comparison of several of these in order to assess which is favoured for removing intact protein whilst retaining peptides. Digest QconCAT™ Protein mTRAQ™ Label Sample (Δ 0 ) Pooled mTRAQ™ Samples (Standards) Monitor by MRM mTRAQ™ Label Sample (Δ 4 ) Enrich Samples for Peptides Plasma + mTRAQ™ (Δ 4 ) Plasma Pool Enriched Sample & mTRAQ™ Δ 0 Peptides Plasma Untreated Plasma Untreated Plasma 10% SDS-PAGE Gel Silver Stain Gel Precipitation 1 vol. Acetone Precipitation 1 vol. Acetone Precipitation 2 vol. Acetone Precipitation 2 vol. Acetone Precipitation 1 vol. Acetonitrile Precipitation 1 vol. Acetonitrile Precipitation 2 vol. Acetonitrile Precipitation 2 vol. Acetonitrile Filtration (5 kDa M W Cut-off) Filtration (5 kDa M W Cut-off) C 8 Extraction (Magnetic Beads) C 8 Extraction (Magnetic Beads) MRM Analysis of peptide recovery A QconCAT™ protein was digested with trypsin and split into two equal aliquots (100 pM each) and each mTRAQ™ labelled. From this, ten sets of standards were made, each containing both sets of mTRAQ™ labelled peptides in an equimolar amount (1 pM). Plasma was re-prepared as described earlier and enriched by the methods showing the most complete protein removal. Following enrichment, the mTRAQ™ (Δ 0 ) standards were added to it (1 pM). The amount of each target peptide lost by enrichment was assessed by MRM analysis by comparing these results back to those of the standards. This is illustrated in Figure 2. Peptide Sequence FiltrationPrecipitation Recovery (%)Variance (%)Recovery (%)Variance (%) AADDEPEYEDGR29.117.527.519.6 AETIGEK5.1127.01.225.9 YEAVPADASSSSEVK65.645.088.918.9 LRPLLEK16.910.1103.818.8 APGGEDEEEGVGGGGGGGELR8.975.722.1122.1 ASLTTPGSEYSSPSVISVSK2.661.1100.117.7 SWLPVVIK0.463.868.60.7 LWIWEK0.2101.6114.916.5 STGSFPFPYVLEPLGASPSETSK1.84.992.718.4 AFYPASLSPPAAGTAASLSTALLR0.660.8105.918.9 AVERAGE13.156.772.627.8