Abundance Abundance Abundance Abundance Abundance Shotgun Proteomics Protein Mixture 1 2 3 MS/MS proteolysis m/z 2 3 Time µlc m/z MS 1 m/z Peptide Mixture m/z
Block Diagram of a Mass Spectrometer Sample In High Vacuum Ion Source Mass Analyzer Detector 8 6 4 2 Relative Abundance1 4 6 8 1 m/z 12 14 16 18 Computer
Block Diagram of a Mass Spectrometer Sample In Tandem Mass Spectrometry or MS/MS High Vacuum Ion Source Mass Analyzer Collisons Mass Analyzer Detector 8 6 4 2 Relative Abundance1 4 6 8 1 m/z 12 14 16 18 Computer
Block Diagram of a Mass Spectrometer Sample In Tandem Mass Spectrometry or MS/MS High Vacuum Ion Source Detector 8 6 4 2 Relative Abundance1 4 6 8 1 m/z 12 14 16 18 Computer
Block Diagram of a Mass Spectrometer Sample In Tandem Mass Spectrometry or MS/MS High Vacuum Ion Source Detector m/z Computer
Abundance Measurement of Peptides by Microcapillary Liquid Chromatography Tandem Mass Spectrometry Microcapillary Chromatography Column Peptide Mixture Peptides are loaded onto the column and separated by liquid chromatography Time
Acquiring Mass Spectrometry Data of Peptides Microcapillary Column 2 kv 3 V Peptides from the column are ionized and transmitted into the vacuum system and trapped in the mass spectrometer
Abundance Acquiring Mass Spectrometry Data of Peptides Peptides from the column are ionized and transmitted into the vacuum system and trapped in the mass spectrometer Peptides are selectively ejected by their mass to produce a mass spectrum The instrument automatically selects a peptide for further analysis Selected m/z m/z
Acquiring a Fragmentation Spectrum The trap is then refilled
Acquiring a Fragmentation Spectrum The trap is then refilled All peptides are ejected from the trap except peptides with the predetermined mass The energy of the isolated peptide is increased resulting in collisions with a gas
Abundance Acquiring a Fragmentation Spectrum The collisions result in structure specific fragments The fragments are then selectively ejected to produce a tandem mass spectrum m/z
Multidimensional Protein Identification Technology (MudPIT) 1 µm i.d. fuse silica Tip is pulled to ~5 μm I.D.
Multidimensional Protein Identification Technology (MudPIT) 1 µm i.d. fuse silica Tip is pulled to ~5 μm I.D. Pack with C18 material 1 st
Multidimensional Protein Identification Technology (MudPIT) 1 µm i.d. fuse silica Tip is pulled to ~5 μm I.D. Pack with C18 material 1 st Pack with SCX material 2 nd
Multidimensional Protein Identification Technology (MudPIT) 1 µm i.d. fuse silica Tip is pulled to ~5 μm I.D. Pack with C18 material 1 st Pack with SCX material 2 nd Peptide digest is loaded offline
Multidimensional Protein Identification Technology (MudPIT) 1 µm i.d. fuse silica Tip is pulled to ~5 μm I.D. Pack with C18 material 1 st Pack with SCX material 2 nd Peptide digest is loaded offline
Multidimensional Protein Identification Technology (MudPIT) H 2 O MeCN NH 4 OAc HPLC ~2 nl/min 2 kv SCX RP- C18 waste
Multidimensional Protein Identification Technology (MudPIT) Step 1 Reverse Phase Gradient H 2 O MeCN NH 4 OAc HPLC 2 kv SCX RP- C18 waste
Multidimensional Protein Identification Technology (MudPIT) H 2 O MeCN NH 4 OAc Step 1 Reverse Phase Gradient Step 2 Salt Pulse HPLC 2 kv SCX RP- C18 waste
Multidimensional Protein Identification Technology (MudPIT) H 2 O MeCN NH 4 OAc Step 1 Reverse Phase Gradient Step 2 Salt Pulse Step 3 Reverse Phase Gradient HPLC 2 kv SCX RP- C18 waste
Multidimensional Protein Identification Technology (MudPIT) H 2 O MeCN NH 4 OAc HPLC Step 1 Reverse Phase Gradient Step 2 Salt Pulse Step 3 Reverse Phase Gradient Step 4 Increase Salt Pulse Repeat 2 kv SCX RP- C18 waste
Relative Abundance Relative Abundance Relative Abundance µlc/µlc/ms/ms of C. elegans Peptides Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative Abundance RT:. - 12. 29.49 34.93 1 95 9 85 8 75 7 65 1 2 3 4 1.61 1.87 11.41 45.53 RT:. - 12.3 37.18 1 37.4 95 9 85 8 75 48.33 48.73 RT:. - 12.2 42.97 1 23.31 95 9 85 8 75 39. 36.26 61.4 RT:. - 12.4 3.79 1 33.7 27.34 7 57.62 21.24 7 35.84 7 37.27 27.5 47.31 65 65 65 35.4 95 9 85 8 75 34.49 46.31 46.8 NL: 4.86E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6154-worm-trizolurea-15n-J1-1 NL: 3.E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6154-worm-trizolurea-15n-J1-2 NL: 3.22E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-3 NL: 3.9E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-4 6 55 5.15 22.9 38.82 4.9 6 55 5 2.86 21.62 37.8 65.92 6 55 5 27.25 6.66 61.28 6 55 5 45 4 35 3 25 2 15 1 5 8.65 12.66 48.81 57.8 3.65 5.73 1 2 3 4 5 6 7 8 9 1 11 64.17 64.5 71.31 65.67 71.71 114.58 77.6 81.56 86.77 111.56 1.89 9.15 11.37 114.88 45 66.11 4 44.58 38.7 71.17 35 57.6 3 63.93 75.92 39.85 25 3.48 6.44 8.17 29.14 2 15 24.33 52.83 84.5 1 84.97 19.83 5.52 2.7 111.47 86.15 18.71 5 4.78 5.82 18.71 88.49 14.88 7.32 14.23 1 2 3 4 5 6 7 8 9 1 11 12 45 4 35 3 25 2 15 1 5 2.24 3.35 49.91 5.54 5.7 57.1 66.73 56.64 96.28 13.31 67.31 76.41 77.6 77.3 78.75 87.44 67.65 6.45 87.61 14.31 97.84 6.13 12.41 19.82 4.32 1 2 3 4 5 6 7 8 9 1 11 12 45 4 35 3 25 2 15 1 5 13.96 29.75 29.29 19.61 24.1 39.24 44.78 44.64 49.41 51.15 56.76 72.16 97.16 9.72 65.3 75.79 96.6 8.6 64.83 98.2 61.79 78.22 98.2 6.34 83.82 93.83 85.68 98.79 3.8 99.26 13.52 11. 1 2 3 4 5 6 7 8 9 1 11 12 71.99 RT:. - 12.2 1 95 9 85 8 75 7 5 6 7 8 42.54 42.83 RT:. - 12.4 1 95 9 85 8 34.47 34.71 41.99 75 75 75 33.94 43.19 7 35.7 7 7 11.36 34.38 RT:. - 12.2 1 95 9 85 8 38.51 41.72 RT:. - 12.1 1 95 9 85 8 48.3 73.23 73.7 89.83 89.47 9.24 88.22 NL: 4.86E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-5 NL: 3.59E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-6 NL: 1.44E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-7 NL: 1.39E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-8 65 6 65 6 4.17 65 6 65 6 36.26 74.16 55 55 55 5.93 69.62 2.35 49.28 52.92 86.86 55 33.33 38.43 38.63 69.93 5 45 33.7 5 45 32.53 4.54 5 45 54.44 69.97 72.76 5 45 11.7 32.77 4.34 4 4 4 56.25 4 17.71 24.19 4.82 35 3 25 2 15 1 5 28.87 37.98 57.48 57.88 44.5 61.29 19.65 26.8 65.47 73.37 9.47 25.9 45.17 56.24 73.88 4.6 18.81 23.89 54.73 67.81 74.13 93.35 9.55 78.16 86.65 94.4 1.45 112.84 35 3 3.97 88.67 25 3.68 11.24 88.49 61.7 2 41.29 56.73 14.73 89.6 88.17 46.97 15 15.3 23.14 54.97 7.92 8.13 73.83 18.36 68.63 76.12 48.25 89.24 68.27 96.65 1 4.8 84.68 62.67 77.12 93.26 1.3 111.27 5 11.87 112.18 113.49 35 3 25 2 15 1 5 56.74 18.23 68.92 6.59 73.21 6.83 73.39 32.28 23.49 24.4 31.29 61.38 42.89 67.11 73.59 79.92 8.25 4.18 81.26 88.1 92.92 93.48 99.3 1.76 112.53 35 3 25 2 15 1 5 19.59 26.71 41.36 49.99 58.79 59.25 58.53 69.36 5.31 59.67 91.17 4.77 93.13 61.6 84.52 94.72 9.18 98.66 18.64 18.2 18.95 1.8 11.93 8.66 8.99 1 2 3 4 5 6 7 8 9 1 11 12 1 2 3 4 5 6 7 8 9 1 11 12 1 2 3 4 5 6 7 8 9 1 11 12 1 2 3 4 5 6 7 8 9 1 11 12 RT:. - 12. 1 95 9 85 8 75 9 1 11 12 3.16 3.32 67.78 9.94 RT:. - 12. 1 95 9 85 8 21.42 RT:. - 12. 1 95 9 85 8 75 75 75 2.95 RT:. - 12.1 1 95 9 85 8 26.72 28.65 NL: 1.55E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-9 NL: 2.25E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-1 NL: 2.45E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6174-worm-trizolurea-15n-H-11 NL: 3.47E7 Base Peak F: ITMS + c ESI Full ms [ 4.-14.] MS 6154-worm-trizolurea-15n-J1-12 7 91.62 7 7 7 65 6 33.55 67.63 65 6 65 6 27.33 65 6 42.56 55 5 45 74.33 55 5 45 66.96 67.19 67.47 55 5 45 55 5 45 2.27 14.93 8.67 42.82 4 35 3 25 2 15 1 5 2.87 21.85 25.97 35.62 4.24 4.66 11.83 48.29 59.53 92.6 18.93 11.65 18.34 1.38 12.12 43.61 48.77 19.67 78.31 18.11 58.64 64.68 12.68 78.69 92.99 19.93 79.2 97.15 3.61 17.62 81.48 98.37 111.2 4.82 113.79 1 2 3 4 5 6 7 8 9 1 11 12 88.63 88.48 92.9 4 35 3 25 2 15 1 5 26.42 35.21 19.24 18.44 29.97 87.91 68.12 89.9 76.36 51.22 51.2 66.9 44.85 55.11 4.66 11.32 63.75 75.64 76.93 63.38 11.31 17.55 11.86 11.8 55.55 69.94 77.53 3.7 19.2 94.42 13.99 112.57 4.3 1 2 3 4 5 6 7 8 9 1 11 12 67.84 19.6 16.92 4 16.57 7.27 35 16.37 31.61 7.52 17.7 3 43.55 43.9 18.4 25 4.51 43.86 18.62 2 44.5 37.92 46.26 85.98 52.64 15.6 18.86 15 72.44 46.45 76.34 76.64 19.68 1 57.1 69.6 86.93 77.1 11.15 9.36 15.88 57.56 64.9 4.7 78.54 91.98 111.37 5 92.61 112.11 1 2 3 4 5 6 7 8 9 1 11 4 35 25.21 62.38 3 11.68 65.97 72.52 25 31.37 39.67 2 111.39 59.61 39.9 56.2 111.2 44.82 77.8 15 8.19 77.68 5.19 1 112.2 6.41 87.14 5 92.32 79.56 93.36 99.44 116.29 5.6 1 2 3 4 5 6 7 8 9 1 11 12 MS/MS Spectra: 281,484 Peptides Identified: 8,13 Proteins Identified With >95% Confidence: 2,346
Humans Insulin Receptor C. elegans DAF-2 IRS1-4 IST-1 pi 3-kinase AGE-1 PDK1 PDK-1 AKT/PKB AKT-1/AKT-2 FKHR FKHRL1 AFX DAF-16
Fractionation to Improve the Coverage of Proteins Involved in Insulin / IGF-1 Signaling Reversed Phase 1 Fractions Soluble Proteins Insoluble Proteins Proteins Identified that are Known to be Involved in the Insulin (NH 4 ) 2 SO 4 Cuts 8 Fractions Density Centrifugation / IGF-1 Signaling Pathway 1 Fractions 1 MudPITs 8 MudPITs 1 MudPITs >6,, MS/MS Spectra, 21,264 Unique Peptide Identifications, and 4,373 Unique Protein Identifications
Targeted Proteomics using nanolc-hsrm-ms
Relative Abundance Relative Abundance Q1 CID Q3 Ion Source 1 521.7 757.6 1 8 8 6 6 4 4 2 2 521. 521.5 522. 522.5 523. 523.5 524. m/z 2 4 6 8 1 m/z
Counts Counts Counts Counts Counts Counts Counts Counts Counts Precursor Ion > Product Ion SRM Chromatograms TGASEAVPSEGK > GK TGSAEAVPSEGK > EGK TGSAEAVPSEGK > SEGK TGSAEAVPSEGK > PSEGK TGSAEAVPSEGK > VPSEGK TGSAEAVPSEGK > AVPSEGK TGSAEAVPSEGK > EAVPSEGK TGSAEAVPSEGK > AEAVPSEGK TGSAEAVPSEGK > SAEAVPSEGK 1x1 5 8x1 4 6x1 4 4x1 4 y2 566.78 > 24.13 2x1 4 33 34 37 38 39 4 32 35 36 2x1 4 1x1 4 y3 9x1 3 6x1 3 566.78 > 333.18 3x1 3 32 33 34 35 36 37 38 39 4 1x1 4 y4 9x1 3 6x1 3 566.78 > 42.21 3x1 3 32 33 34 35 36 37 38 39 4 2x1 4 y5 1x1 4 8x1 3 566.78 > 517.26 4x1 3 32 33 34 35 36 37 38 39 4 1x1 4 y6 8x1 3 5x1 3 566.78 > 616.33 3x1 3 32 33 34 35 36 37 38 39 4 1x1 5 y7 9x1 4 6x1 4 566.78 > 687.37 3x1 4 32 33 34 35 36 37 38 39 4 8x1 3 y8 6x1 3 4x1 3 566.78 > 816.41 2x1 3 32 33 34 35 36 37 38 39 4 3x1 4 2x1 4 4 y9 566.78 > 93.44 1x1 33 34 37 38 39 4 32 35 36 3x1 4 y1 2x1 4 4 566.78 > 131.5 1x1 32 33 34 35 36 37 38 39 4
Synthetic Peptide Standards Relative Abundance Relative Abundance Worm Homogenate 25 µg Relative Abundance Relative Abundance 1 1 8 IDATTHIGGVQIK DAF-16 8 QEDVVIEGWLHK AKT-1 6 6 4 4 2 2 2 21 22 23 24 25 2 21 22 23 24 25 1 8 75 attomole IDATTHIGGVQIK 1 8 7.5 femtomole QEDVVIEGWLHK 6 6 4 4 2 2 2 21 22 23 24 25 2 21 22 23 24 25
Measurement of Absolute Levels of DAF-16 and AKT-1 During Development Using Standard Addition Femtomoles per 25 g Total Protein 5 45 4 35 3 25 2 15 1 5 DAF-16 AKT-1 embryo 18 h 24 h 36 h 48 h 6 h 76 h 8 h 96 h Development Time
Protein of Interest General Approach for Targeted Identification of Proteins by hsrm 1 MAESHLLQWL LLLLPTLCGP GTAAWTTSSL ACAQGPEFWC QSLEQALQCR 51 ALGHCLQEVW GHVGADDLCQ ECEDIVHILN KMAKEAIFQD TMRKFLEQEC 11 NVLPLKLLMP QCNQVLDDYF PLVIDYFQNQ TDSNGICMHL GLCKSRQPEP 151 EQEPGMSDPL PKPLRDPLPD PLLDKLVLPV LPGALQARPG PHTQDLSEQQ 21 FPIPLPYCWL CRALIKRIQA MIPKGALAVA VAQVCRVVPL VAGGICQCLA 251 ERYSVILLDT LLGRMLPQLV CRLVLRCSMD DSAGPRSPTG EWLPRDSECH 31 LCMSVTTQAG NSSEQAIPQA MLQACVGSWL DREKCKQFVE QHTPQLLTLV 351 PRGWDAHTTC QALGVCGTMS SPLQCIHSPD L Any Peptide Identified Previously? (LTQ) YES Approximate Precursor and Product Ions from Previously Acquired Data
Protein of Interest General Approach for Targeted Identification of Proteins by hsrm 1 MAESHLLQWL LLLLPTLCGP GTAAWTTSSL ACAQGPEFWC QSLEQALQCR 51 ALGHCLQEVW GHVGADDLCQ ECEDIVHILN KMAKEAIFQD TMRKFLEQEC 11 NVLPLKLLMP QCNQVLDDYF PLVIDYFQNQ TDSNGICMHL GLCKSRQPEP 151 EQEPGMSDPL PKPLRDPLPD PLLDKLVLPV LPGALQARPG PHTQDLSEQQ 21 FPIPLPYCWL CRALIKRIQA MIPKGALAVA VAQVCRVVPL VAGGICQCLA 251 ERYSVILLDT LLGRMLPQLV CRLVLRCSMD DSAGPRSPTG EWLPRDSECH 31 LCMSVTTQAG NSSEQAIPQA MLQACVGSWL DREKCKQFVE QHTPQLLTLV 351 PRGWDAHTTC QALGVCGTMS SPLQCIHSPD L Any Peptide Identified Previously? (LTQ) YES NO Peptide Candidates Approximate Precursor and Product Ions from Previously Acquired Data Predict Precursor and Product Ions in silico
B2-AR B2-AR Group Means Non-Failing Failing Failing Non-Failing Calsequestrin Calsequestrin Group Means Non-Failing Non-Failing Failing Failing
MLC2 MLC Group Means Non-Failing Failing Failing Non-Failing
Troponin-C: Regulation Protein of Contraction Nonfailing Failing