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

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

  • Slide 1
  • Introduction to 2D LC- MS/MS (Yuanming Luo) Institute of Microbiology Chinese Academy of Sciences
  • Slide 2
  • Fully integrated 2D-LC/ion trap MS
  • Slide 3
  • Hardware Improvement ---- New Orthogonal Ion Source Square Quadrupole New Inter-Octapole Lens New Endcap Electrodes Entrance Lens Attomole Sensitivity !!!
  • Slide 4
  • 1D-strong cation exchange column (Biobasic SCX)
  • Slide 5
  • Pressure cell
  • Slide 6
  • Slide 7
  • Xcalibur- control the instrument
  • Slide 8
  • Bioworks 3.1-database search software package containing SEQUEST
  • Slide 9
  • Application of 2D LC-MS/MS Molecular weight determination 2D gel spots (especially the spots that cant 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)
  • Slide 10
  • Molecular weight determination of myoglobin by BIOMASS Calculation
  • Slide 11
  • Mr:16951.38+ /-0.33
  • Slide 12
  • High throughput gel spot analysis
  • Slide 13
  • Tandem RP Columns
  • Slide 14
  • Automated Protein Identification of 2-D gel spots ? Sensitivity and Throughput !!! SEQUEST Cross-Correlation Comparison Protein identified Digest
  • Slide 15
  • Slide 16
  • High throughput gel spot analysis 1. Protein mixture is separated by 2D gel electrophoresis 2. Excise target gel spot 3. Perform in-gel digestion with trypsin. 4. Extract peptides from gel spot. 5. Run peptide mixture with ProteomeX in 1D High Throughput mode. 6. Data search by TurboSEQUEST software
  • Slide 17
  • ProteomeX Analysis of 2D Gel Spots Using ProteomeX High Throughput Method 65.73 70.49 21.95 45.20 82.59 28.64 19.5341.22 13.77 83.90 100.31 22.68 51.85 79.67 72.35 70.5823.16 35.66 40.14 82.29 22.05 17.20 3.68 95.10 61.76 21.83 51.89 65.7770.2936.7743.07 34.1680.86 82.44 20.55100.61 8.82 0.48 63.93 23.06 29.58 39.32 51.72 79.87 65.88 35.1242.52 59.2017.4482.27 15.18 97.304.34 61.60 21.90 51.85 70.37 48.81 19.6237.34 72.6481.2627.77 12.701.26 98.74 85.70 22.74 51.83 79.86 63.79 70.37 54.56 48.00 29.5681.19 44.47 21.28 11.13 84.87 92.89 8.54 NL: 2.39E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS GelSpot_tPA1_C1 NL: 9.02E6 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa2_c2 NL: 1.16E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa3_c1 NL: 1.41E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa4_c2 NL: 2.11E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa5_c1 NL: 1.15E7 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa6_c2 NL: 8.00E6 Base Peak F: + c Full ms [ 300.00-2000.00] MS gelspot_tpa7_c1 Spot 1 2 3 4 5 6 7 Found t-PA
  • Slide 18
  • Global Protein Identification
  • Slide 19
  • Protein mixtureProtein digests SCX column fractionation Reverse column separation Auto MS/MS detection Tandem MS spectra BioWorks data base search Results
  • Slide 20
  • Plumbing Diagrams for Proteome X. 1D-SCX column 2D-RP1 column 2D-RP2 column
  • Slide 21
  • Global Protein Identification 1. Extract proteins from cell lysates 2. Reduce proteins to peptide fragments by tryptic digestion. 3. Analyze peptide mixture by 2D LC-MS/MS with ProteomeX. 4. Peptide and proteins identified by TurboSEQUEST software.
  • Slide 22
  • Protease Digestion of Proteins
  • Slide 23
  • 1D LC-MS/MS of proteins from A431 cell lysates RT:0.00 - 600.00 050100150200250300350400450500550600 Time (min) 0 50 100 0 50 100 0 50 100 Relative Abundance 0 50 100 0 50 100 33.95 431.84 35.12 1163.80 26.66 652.24 42.92 1138.32 76.43 667.61 68.71 1160.53 50.66 486.92 117.86 563.10 138.63 703.32 148.78 371.00 14.27 344.05 84.72 1154.55 200.37 563.16 17.79 388.10 118.32 431.94 258.97 776.40 140.05 1839.77 228.84 619.48 269.22 444.83 520.91 1912.57 432.24 675.17 8.40 439.73362.07 675.25 341.64 675.16 382.40 675.26 524.05 1511.36 465.75 488.12 294.93 576.29 171.60 1154.56 123.05 926.00 268.84 1285.77 226.50 794.78 12.29 390.90 113.04 897.74 575.63 444.75 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 ng_1029 NL: 4.60E8 Base Peak MS a431_1213_ 8hrg 30 min gradient 60 min 120 min 240 min 480 min
  • Slide 24
  • 2D LC-MS/MS of proteins from A431 cell lysates
  • Slide 25
  • Analysis of proteins from A431 cell lysates 56240min 105480min 44120min 2260min 1630min # of Proteins Identified Gradient 1D 49120 hr. 33710 hr. 1445 hr. # of Proteins Identified Total Run Time 2D
  • Slide 26
  • Yeast Protein separation 20 mM Ammonium chloride, 40 mM Ammonium chloride, 70 mM Ammonium chloride, 100 mM Ammonium chloride,
  • Slide 27
  • Yeast Protein Separation 140 mM Ammonium chloride, 180 mM Ammonium chloride, 220 mM Ammonium chloride,
  • Slide 28
  • Yeast proteins
  • Slide 29
  • Protein # 1708
  • Slide 30
  • 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
  • Slide 31
  • Viewing Results
  • Slide 32
  • Synclein alpha TIC
  • Slide 33
  • Slide 34
  • 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.
  • Slide 35
  • Filters for SEQUEST Results
  • Slide 36
  • On-Line Phosphopeptide Enrichment (IMAC capture)
  • Slide 37
  • Flow Path of an Automated 2D (IMAC + RP)-MS/MS System for the Analysis of Phosphopeptides
  • Slide 38
  • 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.
  • Slide 39
  • Capture of FQ*SEEQQQTEDELQDK Phosphopeptide of -Casein Digest in the 2D LC(IMAC+RP)-MS/MS System Non-phosphorylated peptides flow through IMAC column and captured by and eluted from RP2 Phosphorylated peptide (m/z=1031.7, FQ*SEEQQQTEDELQDK) captured by IMAC column, bound to RP1, and eluted. NL: 1.34E9 NL: 1.80E8 position for m/z=1031.7 on C2 column RP2 column RP1 column
  • Slide 40
  • Neutral Loss Scanning Confirmed the Major Ion at m/z=1031.6 as a P-peptide Neutral loss fragment (-49) M+2H + -49 MS/MS of 1031.6 Phosphorylated peptide (m/z=1031.7, FQ*SEEQQQTEDELQDK)
  • Slide 41
  • Bioworks 3.1 Search Identified the P-peptide with m/z=1031.6 as FQ*SEEQQQTEDELQDK 1+ 2+ (M+2H)-49
  • Slide 42
  • Proteins - Differential Expression (EGF treated and untreated cells) ---- Alternative method for differential analysis of protein expression compared to 2DE strategy
  • Slide 43
  • Protein differential expression 1. Divide A431 cell sample in two: a) Half stimulated by EGF b) Half control 2. Lyse cells 3. Extract proteins from lysates 4. Digest with trypsin 5. Run 2D LC-MS/MS of digests with ProteomeX 6. Proteins identified by TurboSEQUEST software 7. Compare stimulated vs. control
  • Slide 44
  • Automated 2D LC-MS/MS Analysis of Human A431 Cell Proteins 120 mM 10 mM 20 mM 40 mM 60 mM NH 4 Cl 0 mM 80 mM 160 mM
  • Slide 45
  • 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 )
  • Slide 46
  • Proteins Differentially Expressed in Control and EGF- Stimulated A431 Cells
  • Slide 47
  • Proteins Differentially Expressed in Control and EGF-Stimulated A431 Cells (continued) *Only those proteins with two or more peptides identified were compared
  • Slide 48
  • Proteins Identified in Both Control and EGF- Treated A431 Cells
  • Slide 49
  • Proteins Common to Control and EGF-treated A431 Cells (continued)
  • Slide 50
  • Slide 51
  • Differential Protein quantitation -quantitative proteomics
  • Slide 52
  • Stable isotope labeling (SIL) for quanlitative pro