MALDI TOF/TOF technology: applications and workflows for a mass spectrometry research lab

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1 MALDI TOF/TOF technology: applications and workflows for a mass spectrometry research lab Volker Kruft Manager Business Development EMEA, Proteomics Volker.Kruft@eur.appliedbiosystems.com

2 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

3 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

4 The AB SCIEX TOF/TOF Family 4800 MALDI TOF/TOF TM Analyzer The AB SCIEX TOF/TOF TM 5800 System

5 4800 MALDI TOF/TOF System Linear detector Mirror region Source 2 region QuanTIS Precursor Selector Deceleration Stack Laser Camera Reflector detector Sample stage and sample plate OptiBeam on-axis laser Source 1 Region

6 OptiBeam On-Axis Laser The angular distribution of the MALDI ion plume is directed back towards the direction of the incident laser On-axis OptiBeam Laser configuration on the 4800 MALDI TOF/TOF Analyzer and the TOF/TOF 5800 Systems provides optimum MS and MS/MS sensitivity

7 On-Axis vs. Off-Axis Ionization by MALDI y Plume formation before acceleration (v 0 = m/s, = 45 ) Scale enlarged for effect x Trajectory after acceleration Off axis 30 On axis

8 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

9 Proteomics: It s all about time courses Proteome 1 Proteome 2 Proteome 3 Proteome 4 Proteome 5 Proteome 6 Proteome 7

10 The Dynamic Range Challenge: Plasma Proteomics LC/MS range: ~3.5 order of magnitude mg/ml Deplete top 10 proteins *Prostrate specific antigen Cancer marker Where we need to be * ug/ml ng/ml pg/ml 2009 Applied Biosystems N.L. - Confidential Anderson et al.; The human plasma proteome; MCP; 2002

11 Dynamic range in proteomics Courtesy of B. Domon, ETH Zurich

12 The problem of biological variation unrelated to disease Although human genome > 99.9% identical considerable differences in proteome unrelated to disease biomarkers = noise (courtesy of Karl Mechtler, IMP, Vienna)

13 The Do ers in a Cell

14 Importance of MS in the life sciences (as indexed in PubMedLine) MS total MS protein MS environmental MS metabolism MS quantitation MS clinical '06 '08 0 Last updated August 5, 2008

15 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

16 4800 MALDI TOF/TOF Analyzer

% Inte ns ity 5734.6 5839.1 8476.0 11674.7 11728.9 11886.9 4 7 0 0 L i n e a r S p e c # 1 M C = > S M 9 [ B P = 1 1 6 7 4. 2, 1 3 3 ] 16952.4 17171.

17 % Inte ns ity L i n e a r S p e c # 1 M C = > S M 9 [ B P = , ] Praha, March12, 2009 Small proteins linear mode Resolution Thioredoxin: Calibration Mix Resolution Myoglobin: M a s s (m /z )

18 % Intensity Praha, March12, 2009 MSMS MALDI Spectrum of Intact Thioredoxin m/z MS/MS Precursor Spec #1=>NF1.0[BP = , 397] Mass (m/z )

19 4800 MALDI TOF/TOF Analyzer Other Unique Applications: Polymers Fatty Acids Carbohydrates

128.04 155.07 171.07 200.11 217.13 243.14 271.16 112.04 % Intensity 261.17 156.07 186.09 287.19 305.19 331.20 347.19 357.23 375.24 391.24 401.24 419.27 435.26 505.30 519.31 537.30 549.32 565.32 581.

20 % Intensity % Intensity Praha, March12, TOF/TOF: Polymer Fragmentation HO - [CH CH O] 2 2 w O [OCH CH ] - OH 2 2 x [OCH CH ] - OH 2 2 y [OCH CH ] - OH 2 2 z Mass (m/z) MS/MS of sorbitan polyethoxylate (Tween component) MNa with CID no CID Mass (m/z)

21 % Intensity % Intensity S t i t c h e d P S D = > S M 2 5 [ B P = , ] Praha, March12, TOF/TOF: MS/MS of a Triglycerid PSD 3.1E C17H33COOH Mass (m /z) CID-MSMS cleavage of carbon chain: -loss of (C n H 2n+2 ) -loss of (C n H2 n ) -loss of (C n H 2n-2 ) 808 (even) indicates C=C Mass (m/z)

% Intensity Praha, March12, 2009 MSMS of (Man) 7 (GlcNAc) 2 (D3): Mix of Glycosidic Cleavages and Cross Ring Cleavages 100 90 TOF TOF MS/MS Precursor 1582 Spec #1=>NF1.0[BP = 1579.

22 % Intensity Praha, March12, 2009 MSMS of (Man) 7 (GlcNAc) 2 (D3): Mix of Glycosidic Cleavages and Cross Ring Cleavages TOF TOF MS/MS Precursor 1582 Spec #1=>NF1.0[BP = , 1527] -OH M+Na Mass ) Mass (m/z)

23 TOF/TOF: Recent Carbohydrate Publications

24 TOF/TOF: Growing list of publications

25 The Power of LC-MALDI Direct Spotting onto MALDI target TOF/TOF MALDI System omaldi QSTAR Elite Tempo LC MALDI GPS Explorer for Data Analysis MS/MS Analysis and Quantitation (itraq TM ) MS Analysis and Quantitation

% Intensity LC/MALDI result dependent experiment: Optimized selection of precursors for MS/MS

$5 This fraction is used for MS/MS in MALDI 100 90 80 70 60 50 40 y 4 y 6 ESI threshold$ y 22y23 30 y y 27y28 26 20 y y 8 y 5 13 y 29y y 30 20 y y 31 24 10 y y 25 b 2 y y MH + 33 33

y 22y23 30 y y 27y28 26 20 y y 8 y 5 13 y 29y y 30 20 y y 31 24 10 y y 25 b 2 y y MH + 33 33

26 % Intensity LC/MALDI result dependent experiment: Optimized selection of precursors for MS/MS XIC for m/z This fraction is used for MS/MS in MALDI y 4 y 6 ESI threshold LAADEDDDDDDEEDDDEDDDDDDFDDEEAEEKAPVKK y 11 y 12 y 7 y 9 y 10 y 14 y 15 y16 y17 y 18 y 19 y 21 y 22y23 30 y y 27y y y 8 y 5 13 y 29y y y y y y 25 b 2 y y MH b35 b m/z 2008 Applied Biosystems - Confidential Selecting the precursor ion at the optimum elution time affords the highest quality MS/MS spectra

27 % Intensity % Intensity MALDI MS/MS: Dynamic Range ( digging deeper ) 20 sec LC fraction of serum # of monoisotopic peaks: 302 (>S/N 3) Range of intensities: % Selected for MS/MS: 43 most abundant E Mass (m/z) Mass (m/z)

28 % Intensity % Intensity % Intensity % Intensity Praha, March12, 2009 MALDI MS/MS: Dynamic Range ( digging deeper ) Expanded regions MS and MS/MS of well B10 5.3E ,000 counts 380 counts Mass (m/z) Mass (m/z) 100 Corresponding MS/MS of Precursor Ions (Y)FGYSGAFK from transferrin MH E (P)LPPTSAHGNVAEGETKPDPDVTER from beta-1b-glycoprotein MH Mass (m/z) Mass (m/z)

29 Martinsried Contest Tryptic digest of 10,000 cells (human kidney cell line, HEK 293) Dried pellet (about 2mg each) in triplicate (per instrument) How many proteins can you identify?

High Performance Instruments for Robust Protein Identification QSTAR Elite LC/MS/MS System High quality protein identification results in the fastest time

31 High Performance Instruments for Robust Protein Identification QSTAR Elite LC/MS/MS System High quality protein identification results in the fastest time with Smart Exit and up to 7 MS/MS per second MALDI TOF/TOF Analyzer Greatest depth of coverage with intelligent LC MALDI protein identification workflows.

32 Maximizing number of protein identifications when sample is limited: (I) 4800 In-depth protein id in complex samples with a wide dynamic range: Tryptic digest of 10,000 cells (human kidney cell line, HEK 293) LC-MALDI with the 4800 Separation frozen on target plate Complete control in precursor selection without separation time constraints Dynamic Exclusion of precursors: Run 1of 2: 50% of 1 sample Prot ID MALDI MS and MS/MS Run 2 of 2: 50% of 1 sample Prot ID MALDI MS and MS/MS (dynamic exclusion of all precursors from run 1 by mass and retention time)

33 Maximizing number of protein identifications when sample is limited: (II) QSTAR Elite In-depth protein id in complex samples with a wide dynamic range: Tryptic digest of 10,000 cells (human kidney cell line, HEK 293) Optimized IDA runs: Dynamic background substraction Smart CE and Smart Exit Dynamic Exclusion of precursors: Run 1of 2: 50% of 1 sample Prot ID LC/MS/MS Run 2 of 2: 50% of 1 sample Prot ID LC/MS/MS (dynamic exclusion of all precursors from run 1 by mass and retention time)

34 Methods: Copy precursor list plus rentention time Easy in Peak Explorer

35 Method: Paste list into the exclusion list

$Mass (m/z) Praha, March12, 2009 Results: Heat map of the 1 st LC run Heat Map - Demo\Martinsried\LC-02 2h Martinsried 2005 Run #7$

36 Mass (m/z) Praha, March12, 2009 Results: Heat map of the 1 st LC run Heat Map - Demo\Martinsried\LC-02 2h Martinsried 2005 Run #7 Trace: Fraction Number

37 Signal/Noise Praha, March12, 2009 Results: XIC of 1264 Signal/Rauschen XIC /- 0.2 Da not identified RAPFDLFENR heat shock protein DFLLQQTMLR glyoxalase I LWDLTTGTTTR guanine binding protein AFITNIPFDVK ribonucleoprotein Fraction Fraktionsnummer Number

38 Results: # of MSMS and # of proteins 1 st LC: 4276 MSMS 820 proteins (5.2 MSMS = 1 protein) 2 nd LC: 2563 MSMS 430 new proteins (6.0 MSMS = 1 new protein) Get as many good MSMS as possible!!

39 % Inte ns ity Praha, March12, 2009 Peak Selection in LC-MALDI MS fraction 500 (= 81.3 min.) Re fle c tor Spe c #1 M C[BP = , ] MS - Fraktion 500 MS/MS performed in = fr = fr. 499 = fr. 498 = fr = fr = fr Mass (m /z )

Independent False Positive Rate Assessment by Decoy Searching Reverse database searching was used to analyze the false positive rate in protein detection from ProteinPilot Software for each workflow.

40 Independent False Positive Rate Assessment by Decoy Searching Reverse database searching was used to analyze the false positive rate in protein detection from ProteinPilot Software for each workflow. Both ROC curves show good discrimination, specifically both curves have a sharp transition from a nearly vertical segment (correct high scoring peptides) to a nearly horizontal segment (incorrect low scoring peptides). QSTAR Elite System 4800 TOF/TOF Analyzer

41 Proteins Identified by ProteinPilot Software Venn Diagram Comparison of Proteins Found Proteins identified at the 5% aggregate false positive rate Determined from the ROC plot of the ProteinPilot Software results

Comparison of Acquisition Techniques Proteins identified at the 5% aggregate false positive rate Total analysis time - time required to acquire and process the MS/MS

42 Comparison of Acquisition Techniques Proteins identified at the 5% aggregate false positive rate Total analysis time - time required to acquire and process the MS/MS data Processing time was determined by running both datasets on a single processor computer (Dell Latitude D810 laptop with 2.0 GB RAM and 1.86 GHz Pentium M processor).

43 Assessing the Complementarity of ESI and MALDI Two dominant soft ionization strategies LC MALDI has been more flexible due to decoupling of acquisition and LC, however, the technique has been speed limited. Previous work has suggested a complementarities in information Here, we wanted to assess the complementarities vs. random acquisition variation of ESI vs MALDI Sample is universal performance standard E.coli total protein tryptic digest

1% TFA) Tempo LC MALDI Spotting System 720 spots were collected per LC run, 8sec/spot Online

44 LC MALDI Acquisition AB SCIEX TOF/TOF 5800 System Monolithic CapRod column (100 µm ID x 15cm, Merck) 90 minute gradient - 2µL/min (0.1% TFA) Tempo LC MALDI Spotting System 720 spots were collected per LC run, 8sec/spot Online mixing of α-cyano-4- hydroxycinnamic acid matrix (Agilent). TOF/TOF 5800 System 25 most intense precursors per spot Laser frequency of 1000Hz DynamicExit Algorithm

Nano-LC Direct Spotting onto MALDI target LC MALDI Workflow 0kV -2kV pulse -2kV pulse 250msec

36 AB SCIEX TOF/TOF TM 5800 System 1016.64 1150.67 1278.65 1662.83 1456.83 1644.95 1976.15 1948.

76 114 116 113 115 117118 119 121 975.0 1525.2 2075.4 2625.6 3175.8 3726.0 Mass (m/z) 120.

14 857.59 951.64 70.12 551.33 72.12 39.02 983.69 142.12 387.24 486.31 585.35 679.40 957.68 1245.

45 Nano-LC Direct Spotting onto MALDI target LC MALDI Workflow 0kV -2kV pulse -2kV pulse 250msec Protein Pilot Software for Data Analysis AB SCIEX TOF/TOF TM 5800 System Mass (m/z) (A ) Interpretation (A ) Mass (m/z) Mass (m/z)

ESI Orbitrap LC MS Acquisition Reverse phase nanolc was performed using the Agilent 1100 LC system Magic AQ C18 column (75 µm x 100cm column, self packed) at 300 nl/min Same 90 min gradient

46 ESI Orbitrap LC MS Acquisition Reverse phase nanolc was performed using the Agilent 1100 LC system Magic AQ C18 column (75 µm x 100cm column, self packed) at 300 nl/min Same 90 min gradient Acquisition settings used according to vendor specifications *.mgf file was generated using ReAdw4Mascot2.exe, a NIST derivative of the ISB tool, ReAdW for processing the Orbitrap data with ProteinPilot Software Version 3.0

47 Comparing Sample Loading at the Protein Level Online ESI vs LC MALDI Analysis Similar protein ID rates achieved at 20ng More proteins found by LC MALDI at 100 and 500 ng Acquisition time was 2.5x longer

48 Comparing Sample Loading at the Peptide Level Online ESI vs LC MALDI Analysis Similar peptide ID rates achieved at 20ng More peptides found by LC MALDI at 100 and 500 ng Acquisition time was 2.5x longer

49 Protein / Peptide Intersection 500 ng Loading Level Comparison of Single Best Replicates ESI with MALDI acquisitions appear complementary and suggest greater depth of coverage from combination than a signal technique alone. But how similar are replicates from the same instrument? 5800 system 700 proteins Orbitrap 544 proteins 5800 System 4710 peptides Orbitrap 2259 peptides (30.1%) (59.9%) (10.0%) (15.3%) (59.4%) (25.3%)

Peptide Level Intersection of Replicates Average intersection at the peptide level between 500 ng replicates: For 5800 system: 64% (Sample 1-3) For

50 Peptide Level Intersection of Replicates Average intersection at the peptide level between 500 ng replicates: For 5800 system: 64% (Sample 1-3) For Orbitrap: 61% (Samples 4-6) Between instruments: 25% (red boxes) This clearly shows that intra-instrument replicates are more similar than inter-instrument replicates

51 Protein Level Intersection of Replicates Average intersection at the protein level between 500 ng replicates: For 5800 system: 78% (Sample 1-3) For Orbitrap: 74% (Samples 4-6) Between instruments: 60% (red boxes) Even at the protein level, differences are apparent

52 Comparison of Protein Identification Quality ProteinPilot Software Results from Orbitrap and 5800 System Average Unused ProtScores for all proteins were aligned from each instrument (500 ng replicates). Points above the diagonal mean those proteins have more peptides and therefore get better Protein scores with 5800 system. The clumps at the lowest limits are proteins not found at all by one instrument or the other.

Reproducibility of LC MALDI Acquisition LC MALDI is a unique acquisition strategy as the MS is decoupled from the chromatographic separation, enabling as much or as little time to be taken during MS

53 Reproducibility of LC MALDI Acquisition LC MALDI is a unique acquisition strategy as the MS is decoupled from the chromatographic separation, enabling as much or as little time to be taken during MS and MS/MS acquisition Intelligent precursor selection (using MS peak apex selection) and intelligent 72 MS/MS acquisition Rep3 751 (DynamicExit Algorithm) enable the most information to be obtained from each sample spot in an LC MALDI run 44 Rep % ~ 5% global protein level used for Venn Rep2 719

54 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

55 Experimental Approaches to Biomarker Discovery (Relative and Absolute Quantitation) Profiling approaches (not MS-based): - gel-based, e.g. DIGE - SELDI - chromatography Non-tagging approaches (MS-based): - peptide count ( protein abundance index ) - ion-current-based (XIC) Tagging/labeling (MS-based): - metabolic stable isotope labeling (SILAC, 15 N) - enzymatic stable isotope labeling ( 18 O incorporation during digestion) - chemical stable isotope labeling (cicat, itraq, ICPL, mcat)

56 Advantages of Stable Isotope Labeling simultaneous quant and ID in a single run flexibility in fractionation strategy (not limited to 1D LC) high protein coverage reproducibility of sample prep across samples multiplexing possible: multiple sample comparisons in a single run, reduction in analysis time simple and easy to use

57 Characteristics of a good tagging chemistry induces mass shift by stable isotopes (not deuterium) low molecular weight reporters in a quiet region in MS/MS

58 Summed Ion Intensity Praha, March12, 2009 Reporter Group Placement: Selection of Quiet Region Summed Ion Intensity (~75,000 Spectra) m/z

59 Characteristics of a good tagging chemistry induces mass shift by stable isotopes (not deuterium) low molecular weight reporters in a quiet region in MS/MS multiplexing isobaric simple and easy to use

Parallel Denature & Digest Praha, March12, 2009 itraq Workflow Isobaric Tag Total mass = 145 Amine specific peptide reactive group (NHS) O Reporter Group mass 114 117 (Retains Charge) N N O O N

60 Parallel Denature & Digest Praha, March12, 2009 itraq Workflow Isobaric Tag Total mass = 145 Amine specific peptide reactive group (NHS) O Reporter Group mass (Retains Charge) N N O O N Balance Group Mass (Neutral loss) O (Ross et. al, MCP 3, , 2004) PRG + S1 S2 S PRG + -PRG + Mix MS N H N H N H -N H b 114 b 115 b MS/MS b 117 y y y y S PRG +

61 Parallel Denature & Digest Praha, March12, 2009 itraq Workflow PRG + S1 S2 S PRG + -PRG + Mix MS N H N H N H -N H b 114 b 115 b MS/MS b 117 y y y y S PRG MS: intact labelled peptides are identical, signal intensities are additive! Mass (m/z)

62 Parallel Denature & Digest Praha, March12, PRG + S1 S2 S PRG + -PRG + Mix MS N H N H N H -N H b 114 b 115 b MS/MS b 117 y y y y S PRG + MS/MS: y- and b-ions are additve from 4 individual samples!

63 *VLVDTDYK* MS/MS: reporter ions quantitate 4 individual samples!

64 The itraq workflow itraq - Isobaric Tags for Rel. and Abs. Quantification Balance: H 3 C N N O O N O NH 2 - reactive group: NHS O Reporter: X 3 C O O N O 4 isobaric tags: 145 Da each O X X From: Ross O PL et al. Ross PL et al. MCP 3(12), 1154ff (2004) and Lenčo J, personal communication O N O

65 The itraq workflow itraq - Isobaric Tags for Rel. and Abs. Quantification 114 N H 3 C N O O Peptid 1 MSMS Fragment s m/z N H 3 C N O O Peptid 2 MSMS MS Fragment s m/z MSMS 116 N H 3 C N O O Peptid 3 MSMS Fragment s m/z N H 3 C N O O Peptid 4 MSMS Fragment s m/z 117.1

66 Selected itraq Applications Discovery and Identification of Protein Markers in Endometrial Carcinoma Study of Deregulated Signaling Pathways in Cancer (c-kit/kdr kinase dual inhibitor) Discovery of Putative Biomarkers for Anaplastic Oligodendrogliomas in Human Brain Biomarker Discovery in Human Cerebral Spinal Fluid of Patients with Various Neurodegenerative Diseases Studies on the Composition of Synapsis in Mouse Brain

67 Selected itraq Applications (submitted or in press)

68 Selected itraq Applications Quantitation of Natural Peptidomes Quantitation of Amino-Phospholipids in Tissue Quantitation of Non-Enzymatic Glycosylation of Milk Products Localization of Proteins in the Plant Golgi Current itraq applications are limited by our lack of imagination rather than the itraq technology (Phil Andrews)

itraq Reagents 8-plex Chemistry Isobaric Tag: Total mass = 305 Reporter Group O N Balance Group N O O N Peptide Reactive Group O (PRG) 113 114 115 116 N N N 13 CH 2 N 13 CH 2 15 N+ N+ N+ 15 N+ 13

69 itraq Reagents 8-plex Chemistry Isobaric Tag: Total mass = 305 Reporter Group O N Balance Group N O O N Peptide Reactive Group O (PRG) N N N 13 CH 2 N 13 CH 2 15 N+ N+ N+ 15 N+ 13 CH 2 13 CH CH 3 13 CH 3 13 CH 3 N 15 N 15 N 15 N 13 CH 2 13 CH 2 13 CH 2 13 CH 2 13 CH 2 13 CH 2 15 N+ 13 CH 2 15 N+ 13 CH 2 15 N+ 13 CH 2 15 N+ 13 CH 2 13 CH 2 13 CH 2 13 CH 2

70 % Intensity Praha, March12, 2009 Glu-Fib peptide 8plex MS MS/MS Mass (m/z) y9(+1) 1.1E y6(+1) Reporter ions y13(+1) y1(+1) b2(+1) y7(+1) 20 b3(+1) 10 V E R y2(+1) b6(+1) y4(+1) b4(+1) y8(+1) b5(+1) a8(+1) y14(+1) b14(+1) y15(+1) Mass (m/z) A B. R (A ) F (A ) R F Mass (m/z) Expected Ratio: 1:1:1:1:1:1:1: (A162.86) Mass (m/z) Expected Ratio: 1:2:2:5:5:2:1: (A199.04)

71 Seminar Outline but no time. ;-))) Examples of itraq applications 1) organellar proteomics, protein location 2) signaling pathways, c-kit/kdr inhibitor 3) cell cycle proteomics, Caulobacter cresentus 4) ocular melanoma, protein quant in vitreous fluid 5) Fragile X, protein quant in hippocampal synapses 6) Alzheimer, protein quant in CSF

72 150 publications

73 Seminar Outline TOF/TOF family: technology overview Protein analysis challenges Intelligent protein identification Quantitative proteomics MSI: Mass Spectrometric Imaging

74 Mount Sample Slice Tissue Sections Data Processing Spray Coat with Matrix Acquisition Software 4800 MALDI TOF/TOF Analyzer Load Sample omaldi QSTAR Elite

75 Total Solution MS Imaging Package MALDI Imaging Starter kit for the 4800 System from Laser Biolabs Leap Sprayer from Leap Technologies MSI Imaging data acquisition tool TissueView 1.0 Software MarkerView Software for statistical analysis

76 Tissue Imaging TissueView Software Applications: - Extracted Ion Image (EII) - Regions of Interest (ROI)

78 Mass Spectral Images : Whole Rat Body (head) -TOF MS imaging m/z + =616 Optical Image m/z + =798 Ion Intensity: Low High

79 Overlaying Multiple Extracted Ion Images Red m/z ~ 796 Green m/z ~ 635 Blue m/z ~ 867

80 TissueView Software R G Overlay up to 3 cluster images: B Complementary nature of different clusters apparent

81 TissueView Software: Calculate statistics of region of interest, providing the average intensity of the mass imaged in the region of interest Export images as.tiff or.jpeg images, or simply copy and paste into another program

82 MALDI MSI covering a wide mass range 18 kda 2510 Da 2028 Da 1540 Da 21.8 kda 4618 Da 1909 Da optical image

83 Whole rat body TOFMS image of compound Q (462 Da) -some background interference observed

84 MSMS analysis eliminates Background interference Parent at 462 Da MS/MS fragment at 168 Da

85 Use of on-tissue trypsin digest to aid identification

86 Use of on-tissue trypsin digest to aid identification

87 High Resolution MSI: Oversampling Perceived limitation of MSI for spatial resolution is laser spot size Jurchen et. al recently described oversampling technique that increases spatial resolution without requiring changes to MALDI laser Using this technique, resolution can be increased beyond the laser spot size 50 µm Burnt-Out Spot Fresh Sample (25 µm width) 50 µm Sample ~500 ms Shift Plate 25 µm Laser Spot Burnt-Out Spot

periodic noise removing/fft filtering, prior to

89 Outlook: Advanced data processing (research grade) (spectrum normalization, exclusion of margin spectra, periodic noise removing/fft filtering, prior to clustering and visualization) Normalization: peptida ca. 7 kda before after

90 Conclusions protein research/proteomics is an interesting field to be in MALDI TOF/TOF technology offers the most versatile applications in protein ID MALDI and ESI are complementary

91 but beware: not every TOF/TOF is a TOF/TOF... Picture from

92 Trademarks & Licensing itraq, MIDAS and TEMPO are trademarks and QTRAP, QSTAR and MALDI TOF/TOF are registered trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applera Corporation and MDS Inc. Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. All other trademarks are the property of their respective owners. For Research Use Only. Not for use in diagnostic procedures Applera Corporation and MDS Inc. All rights reserved. Information subject to change without notice.

93 감사 Děkuji Paldies Tapadh leibh Thanks ありがとう谢谢 Hvala Благодаря! Tak Niżżik ħajr Спасибо Obrigado Tesekkurler Danke Salamat po cảm ơn Thank you for your attention Ačiu Gracias Kiitos Dziękuję дєкую Гялайлаа Köszönöm 감사 Dank Merci Mulţumesc Eu ristw Tack Děkuij Grazie ( Þakka þér

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow

Multiplex Protein Quantitation using itraq Reagents in a Gel-Based Workflow Purpose Described herein is a workflow that combines the isobaric tagging reagents, itraq Reagents, with the separation power