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Introduction to 2D LC-MS/MS

Introduction to 2D LC-MS/MS. ( Yuanming Luo) Institute of Microbiology Chinese Academy of Sciences. Fully integrated 2D-LC/ion trap MS. Hardware Improvement ---- New Orthogonal Ion Source. New Endcap Electrodes. Entrance Lens. Square Quadrupole. New Inter-Octapole Lens.

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Introduction to 2D LC-MS/MS

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  1. Introduction to 2D LC-MS/MS (Yuanming Luo) Institute of Microbiology Chinese Academy of Sciences

  2. Fully integrated 2D-LC/ion trap MS

  3. Hardware Improvement ---- New Orthogonal Ion Source New Endcap Electrodes Entrance Lens Square Quadrupole New Inter-Octapole Lens Attomole Sensitivity !!!

  4. 1D-strong cation exchange column (Biobasic SCX)

  5. Pressure cell

  6. Xcalibur-control the instrument

  7. Bioworks 3.1-database search software package containing SEQUEST

  8. Application of 2D LC-MS/MS • Molecular weight determination • 2D gel spots (especially the spots that can’t be identified by PMF analysis) • Protein complex (after primary factionation) • Proteome separation and identification • Multi-dimensional liquid chromatography MS-based differential proteomics • Quantitative proteomics (including ICAT or stable isotope labeling-based differential proteome analysis) • Analysis of posttranslational modification (data dependent)

  9. Molecular weight determination of myoglobin by BIOMASS Calculation

  10. Mr:16951.38+/-0.33

  11. High throughput gel spot analysis

  12. Tandem RP Columns

  13. Sensitivity and Throughput !!! Digest ? SEQUEST Cross-Correlation Comparison Protein identified Automated Protein Identification of 2-D gel spots

  14. High throughput gel spot analysis • Protein mixture is separated by 2D gel electrophoresis • Excise target gel spot • Perform in-gel digestion with trypsin. • Extract peptides from gel spot. • Run peptide mixture with ProteomeX in 1D High Throughput mode. • Data search by TurboSEQUEST software

  15. RT: 0.00 - 102.10 69.67 100 74.38 50 51.42 22.49 75.40 66.11 28.29 42.27 13.98 82.75 10.92 97.56 0 61.66 100 51.90 50 0 100 50 0 100 Relative Abundance 50 0 100 50 0 100 50 0 100 50 0 0 2 4 6 8 10 12 14 16 18 20 Time (min) Analysis of 2D Gel Spots Using ProteomeX High Throughput Method Spot 1 Found t-PA NL: 2.39E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS GelSpot_tPA1_C1 NL: 9.02E6 2 Base Peak F: + c Full ms [ 65.73 70.49 21.95 300.00-2000.00] MS 45.20 82.59 28.64 19.53 41.22 13.77 83.90 100.31 gelspot_tpa2_c2 Found t-PA NL: 1.16E7 22.68 3 Base Peak F: + c Full ms [ 51.85 79.67 300.00-2000.00] MS 72.35 23.16 70.58 35.66 40.14 82.29 gelspot_tpa3_c1 22.05 17.20 3.68 95.10 NL: 1.41E7 61.76 4 Base Peak F: + c Full ms [ 21.83 300.00-2000.00] MS 51.89 gelspot_tpa4_c2 36.77 43.07 65.77 70.29 34.16 80.86 82.44 20.55 100.61 8.82 0.48 NL: 2.11E7 Found t-PA 63.93 5 23.06 Base Peak F: + c Full ms [ 300.00-2000.00] MS 29.58 39.32 51.72 gelspot_tpa5_c1 79.87 65.88 35.12 42.52 17.44 59.20 82.27 15.18 4.34 97.30 NL: 1.15E7 61.60 6 Base Peak F: + c Full ms [ 21.90 300.00-2000.00] MS 51.85 70.37 48.81 19.62 37.34 27.77 72.64 81.26 gelspot_tpa6_c2 1.26 12.70 98.74 85.70 7 NL: 8.00E6 22.74 51.83 79.86 63.79 Base Peak F: + c Full ms [ 70.37 54.56 300.00-2000.00] MS 48.00 29.56 81.19 44.47 21.28 11.13 84.87 92.89 8.54 gelspot_tpa7_c1

  16. Global Protein Identification

  17. Global Protein Identification Reverse column separation SCX column fractionation Protein mixture Protein digests Auto MS/MS detection Results Tandem MS spectra BioWorks data base search

  18. Plumbing Diagrams for Proteome X. 2D-RP2 column 1D-SCX column 2D-RP1 column

  19. Global Protein Identification • Extract proteins from cell lysates • Reduce proteins to peptide fragments by tryptic digestion. • Analyze peptide mixture by 2D LC-MS/MS with ProteomeX. • Peptide and proteins identified by TurboSEQUEST software.

  20. Protease Digestion of Proteins

  21. NL: 2.28E9 Base Peak MS A431_30min G_1029 NL: 2.65E9 Base Peak MS a431_60min g_1029 NL: 1.28E9 Base Peak MS a431_120mi ng_1029 NL: 1.49E9 Base Peak MS a431_240mi 258.97 140.05 228.84 ng_1029 776.40 1839.77 269.22 619.48 444.83 NL: 520.91 432.24 4.60E8 8.40 1912.57 675.17 Base Peak 439.73 362.07 341.64 382.40 524.05 MS 675.25 465.75 675.16 675.26 a431_1213_ 1511.36 294.93 171.60 488.12 123.05 268.84 8hrg 226.50 12.29 113.04 576.29 575.63 1154.56 926.00 1285.77 794.78 390.90 897.74 444.75 RT: 0.00 - 600.00 33.95 431.84 100 35.12 50 1163.80 0 26.66 652.24 100 42.92 1138.32 76.43 50 667.61 0 68.71 50.66 1160.53 100 486.92 117.86 138.63 563.10 Relative Abundance 703.32 50 148.78 371.00 0 14.27 84.72 200.37 344.05 100 1154.55 563.16 17.79 118.32 388.10 431.94 50 0 100 50 0 0 50 100 150 200 250 300 350 400 450 500 550 600 Time (min) 1D LC-MS/MS of proteins from A431 cell lysates 30 min gradient 60 min 120 min 240 min 480 min

  22. 2D LC-MS/MS of proteins from A431 cell lysates

  23. Gradient 1D # of Proteins Identified Total Run Time 2D # of Proteins Identified 30min 16 5 hr. 144 60min 22 120min 44 10 hr. 337 20 hr. 491 240min 56 480min 105 Analysis of proteins from A431 cell lysates

  24. Yeast Protein separation 20 mM Ammonium chloride, 40 mM Ammonium chloride, 70 mM Ammonium chloride, 100 mM Ammonium chloride,

  25. Yeast Protein Separation 140 mM Ammonium chloride, 180 mM Ammonium chloride, 220 mM Ammonium chloride,

  26. Yeast proteins

  27. Yeast proteins Protein # 1708

  28. 2D LC-MS/MS of Yeast proteins • Time: 15 hours • Gradient: 5 – 65% Acetonitrile in 2 hrs in each step • Proteins searched by • Bioworks 3.1 • Proteins identified: 1708 • Throughput: 113.8 proteins/hr

  29. Viewing Results

  30. TIC Synclein alpha

  31. Filters for SEQUEST Results • Xcorr：+1>1.5, +2>2.0, +3>2.5 • ∆CN: >0.1 • When three or fewer peptides for an individual protein passed the criteria (1) the spectrum quality (S/N, match rate) (2) some continuity must be present among the b or y fragments (3) if proline is predicted to be present, then the corresponding y fragment should give an intense peak. (4) unidentified intense peaks should be verified as being either doubly charged.

  32. Filters for SEQUEST Results

  33. On-Line Phosphopeptide Enrichment (IMAC capture)

  34. 1D-IMAC Column 10 –port valve in mass spectrometer Injector Sample loop Sample loop IMAC 2 2 2 2 2 SCX RP2 1 1 1 1 1 RP 1 LCQ Deca XP Plus- mass spectrometer 3 3 3 3 3 Pump 1 Pump 1 Pump 1 Column 1 Sample valve injector injector injector 6 6 6 6 6 4 4 4 4 4 5 5 5 5 5 To Waste Sample Pump Analytical Pump Flow Path of an Automated 2D (IMAC + RP)-MS/MS System for the Analysis of Phosphopeptides

  35. Procedure Used for Automated 2D LC(IMAC+RP)- MS/MS Analysis of Phosphopeptides Step 1: Load IMAC column Step 2: Load peptides on IMAC column. Flow-through peptides captured by RP2 column. Step 3: Wash IMAC column. The bound peptides are then eluted by phosphate buffer on to RP1, while the flow-through peptides trapped on RP2 are being analyzed by LC/MS. Step 4: The bound phosphopeptides on RP1 are analyzed by LC/MS/MS.

  36. Non-phosphorylated peptides flow through IMAC column and captured by and eluted from RP2 NL: 1.34E9 RP2 column position for m/z=1031.7 on C2 column Phosphorylated peptide (m/z=1031.7, FQ*SEEQQQTEDELQDK) captured by IMAC column, bound to RP1, and eluted. NL: 1.80E8 RP1 column Capture of FQ*SEEQQQTEDELQDK Phosphopeptide of -Casein Digest in the 2D LC(IMAC+RP)-MS/MS System

  37. Neutral Loss Scanning Confirmed the Major Ion at m/z=1031.6 as a P-peptide Neutral loss fragment (-49) MS/MS of 1031.6 M+2H+-49 Phosphorylated peptide (m/z=1031.7, FQ*SEEQQQTEDELQDK)

  38. Bioworks 3.1 Search Identified the P-peptide with m/z=1031.6 as FQ*SEEQQQTEDELQDK (M+2H)-49 (M+2H)-49 2+ 1+

  39. Proteins - Differential Expression (EGF treated and untreated cells) ----Alternative method for differential analysis of protein expression compared to 2DE strategy

  40. Protein differential expression • Divide A431 cell sample in two: • Half stimulated by EGF • Half control • Lyse cells • Extract proteins from lysates • Digest with trypsin • Run 2D LC-MS/MS of digests with ProteomeX • Proteins identified by TurboSEQUEST software • Compare “stimulated” vs. “control”

  41. 60 mM NH4Cl 0 mM 10 mM 80 mM 20 mM 120 mM 40 mM 160 mM Automated 2D LC-MS/MS Analysis of Human A431 Cell Proteins

  42. Automated 2D-LC-LC/MS-MS Analysis of Human A431 Cell Proteins(continued) 200mM 300mM 500mM 900mM Total Proteins Identified= 709, using Bioworks 3.1 with TurboSequest (Xcorr = 1.5, 2.0, and 3 for charge states +1, +2, and +3, respectively)

  43. Proteins Differentially Expressed in Control and EGF-Stimulated A431 Cells

  44. Proteins Differentially Expressed in Control and EGF-Stimulated A431 Cells (continued) *Only those proteins with two or more peptides identified were compared

  45. Proteins Identified in Both Control and EGF-Treated A431 Cells

  46. Proteins Common to Control and EGF-treated A431 Cells (continued)

  47. Proteins Common to Control and EGF-treated A431 Cells (continued)

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