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ICAT TM Reagent Based Differential Protein Expression Analysis Using MALDI TOF Tandem MS

ICAT TM Reagent Based Differential Protein Expression Analysis Using MALDI TOF Tandem MS. Xiangping Zhu, Armin Graber, Steve Hattan, Nikita Khainovski, Jason Marchese, Steve Martin, Dale Patterson, Babu Purkayastha, Anthony Romeo, Brian Williamson, Peter Juhasz. Applied Biosystems

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ICAT TM Reagent Based Differential Protein Expression Analysis Using MALDI TOF Tandem MS

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  1. ICATTM Reagent Based Differential Protein Expression Analysis Using MALDI TOF Tandem MS Xiangping Zhu, Armin Graber, Steve Hattan, Nikita Khainovski, Jason Marchese, Steve Martin, Dale Patterson, Babu Purkayastha, Anthony Romeo, Brian Williamson, Peter Juhasz Applied Biosystems Framingham, Massachusetts

  2. Outline of the Presentation • MDLC-MALDI tandem mass spectrometry • for protein identification and quantification • ICATTM cleavable reagent to enhance • protein quantification and identification • Application of MDLC-MALDI-TOF-MS/MS • and ICAT cleavable reagent in nonsense- • mediated mRNA decay (NMD) of yeast • proteome

  3. Digest Proteins In-solution MH+ Strong Cation Exchange LC 100 7834.5 y6 80 1092.57 Identification 60 100 895.3 1291.68 80 40 PGV 939.58 y3 y4 % Intensity y7 1108.58 60 % Intensity y8 1654.94 833.45 20 V b2 b3 b4 b7 y9 1384.80 F 40 1044.59 MSMS 1820.02 1506.82 0 20 61 275 489 703 917 1131 0 Mass (m/z) 802.0 1068.4 1334.8 1601.2 1867.6 2134.0 Mass (m/z) MS Reversed phase nano-LC and spotting samples on MALDI plates A Possible Workflow Based on Off-line Coupling of MALDI-TOF-MS/MS to MDLC

  4. Attributes of Off-line Coupling of MALDI to LC • MALDI analysis can be faster or slower than LC separation • Sample persistence • More sophisticated experimental workflows can be designed

  5. Quantification with ICAT Pairs r = 0.9997 18 2.5 sec 16 1.2 14 12 RSD % 0.4 10 log(H/L) 8 -2.2 -1.2 -0.2 0.8 -0.6 6 4 2 -1.6 0 0 5 10 15 20 25 log(heavy ICAT reagent peptide conc. (nmol/mL)) Number of Acquisition Positions (50 shots each position) Quantification of Peptides with MALDI-MS and Isotope-coded Affinity Tag Reagents Measurement of ICATTM Reagent Peptide Pair Ratio (H/L 1:10) n = 6 • Relative standard deviation of peptide ICAT reagent pair ratio is • smaller than 5% with 500 laser shots or more • Linearity maintains with multiple orders of magnitude

  6. ICAT Cleavable Reagent 227 (236) amu Biotin (C10H17N3O3) I Cleavable Linker Label 9 x 12C/13C Requirements of ICATTM Reagent in Quantification and Identification of Proteins by MDLC-MALDI-TOF-MS/MS • Coelution of light and heavy ICAT Reagent peptides from LC • Enhancement of peptide fragmentation See Brian Williamson’s Poster WPA-023 for Detailed ICAT Cleavable Reagent

  7. 100 4380 100 1694.96 4380 % Intensity 1703.99 % Intensity 1694.97 160 1704.01 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 Mass (m/z) 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 Mass (m/z) 80 100 1694.90 4380 100 4380 1703.93 % Intensity 0 % Intensity 72 73 74 75 76 77 78 79 1694.96 Fraction Number 1703.98 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 Mass (m/z) 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 Light Mass (m/z) Heavy 1694.93 100 4380 100 4380 1703.96 % Intensity % Intensity 1694.93 1703.96 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 0 1691.0 1695.4 1699.8 1704.2 1708.6 1713.0 Mass (m/z) Mass (m/z) 12C/ 13C ICATTM Reagent Labeled Peptide Pairs Co-elute from Reversed Phase LC Ratio H/L = 0.63 Ratio H/L = 0.63 #73 #76 Ratio H/L = 0.63 Ratio H/L = 0.64 #74 #77 Ratio H/L = 0.66 Ratio H/L = 0.60 #75 #78 Average = 0.63 Standard Deviation = 0.020

  8. SLHTLFGDELCK ICAT b3 H 100 MH+ 90 80 70 b4 % Intensity 60 y6 50 HT y7 y2 40 269 y10 b9 b6 b10 y9 y8 b3 – 18 b8 30 y2 - 17 y4 y3 HT- 18 20 b11 y5 L a6 F 10 0 80.0 398.4 716.8 1035.2 1353.6 1672.0 Mass (m/z) Fragmentation of Light ICATTM Cleavable Reagent Labeled Peptide

  9. Saccharomyces cerevisiae Strains Wild Type Strain HFY1200 Nonsense-mediated mRNA Decay Eliminating mRNAs containing premature nonsense codons Minimize Truncated Polypeptides Mutant Strain HFY870 Mutation in UPF1 gene Stabilizing mRNAs containing premature nonsense codons Accumulate Truncated Polypeptides A large number of genes involved in metabolism, telomere maintenance, cell rescue/defense, and cell growth and development, are up/down regulated by >2x in NMD2

  10. SCX on Vision Avidin & Cleavage Nano-LC 2 3 4 Collect 35 fractions with gradient Isolate ICAT reagent labeled peptides and cleave with acid Separation/ Spotting with Probot Submitting to Data Processing Software MS and MS-MS on AB 4700 5 6 Analytical Approach of Yeast Samples Label and Digestion 1 Label WT yeast proteins with light ICATTM reagent and MT proteins with heavy ICAT reagent . Combine two samples and do digestion in-solution (100 mg)

  11. Expression Dependent Selection of Precursorsfor MS-MS  = 0.17 Average = 1.22 non- differentially expressed differentially expressed Features of precursor selection • expression dependent • light only/heavy only • /more abundant • singlets enabled/disabled • dynamic exclusion • exclusion lists • adduct exclusion

  12. 12C/ 13C Pair Ratio 1379.8 0.72 2128.1 0.73 1601.02 100 1715.7 90 80 b2 MH+ 100 70 100 1610.04 90 60 90 2128.19 % Intensity 80 50 80 70 % Intensity 40 70 b3 2119.18 60 30 y7 60 y3 y5 50 20 y9 y11 2055.17 1401.95 % Intensity 50 1474.90 1714.05 y6 40 1597.99 y13 10 2053.19 1781.00 1272.75 1538.93 1975.11 2113.25 1373.87 b5 F,a1(+1) 40 30 0 y8 y12 y10 1156.0 1376.2 1596.4 1816.6 2036.8 2257.0 SQ b6 Y b4 20 30 Mass (m/z) y15 I y14 b2(+1) 10 20 b3(+1) 0 52.0 436.8 821.6 1206.4 1591.2 1976.0 10 y7(+1) y11(+1) y5(+1) Mass (m/z) y9(+1) y3(+1) F,a1(+1) y12(+1) y8(+1) y10(+1) 348.40 y2(+1) b6(+1) 216.28 Q,K y15(+1) I 1495.40 y14(+1) 0 10.0 455.4 900.8 1346.2 1791.6 2237.0 Quantification and Identification of Yeast Proteins Identification by MSMS Quantification by MS Mass (m/z) Average 0.72

  13. Data Processing Workflow • Quantification of ICATTM reagent labeled peptide pairs by MS data • Precursor selection for MSMS • Pairs with ratios having significant deviation from average • Singlets • Pairs with ratios not having significant deviation from average • Identification of proteins by database searching using MSMS data • Parsing results into an Oracle database • Reduce protein list to a non-redundant set • Link identities to quantitative information

  14. Differentially Expressed Proteins from SCX Fraction 7 out of 35 Fractions

  15. 35% 80 30% 70 25% 60 20% 50 40 15% protein count 30 10% 20 5% 10 0% 1 1 2 2 3 3 4 4 5 5 6 6 0 1 2 5 0.2 0.6 0.8 1.2 1.4 1.6 1.8 2.3 2.5 2.7 3.2 3.8 4.8 6.2 ICAT ratio (light/heavy) Distribution of 480 Unique NMD2 Yeast Proteins Labeled with ICATTM Reagent Distribution of Differentially Expressed Proteins in NMD2 Knock- out Yeast Mean = 1.206 Standard deviation = 0.293 Proteins # of standard deviations from mean

  16. Summary • MDLC-MALDI-TOF-MS/MS and ICATTM cleavable reagent approach is powerful for protein identification and quantification, especially for mapping differential protein expression • Sophisticated experiments are facilitated by off-line coupling MALDI-TOF-MS/MS to peptide separation

  17. Applied Biosystems Scott Daniels Sasi Pillai Nicholas Paschalides Ioannis Papayannopoulos Sau-Mei Leung University of Massachusetts Allan Jasobson Feng He Acknowledgements ICAT is a trademark of the University of Washington exclusively licensed to Applied Biosystems Group of Applera Corporation Mascot is a registered trademark of Matrix Science LTD

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