An Overview of Recent Developments for Mass Spectrometry in the Field of Proteomics

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1 An Overview of Recent Developments for Mass Spectrometry in the Field of Proteomics Jeffrey W. Finch, Ph.D. 44th Rocky Mountain Conference July 30th, 2002

2 Outline 1. Background a. Proteomics b. MS Tools c. Challenges for chemical analysis by MS 2. Sample preparation a. Solubilization factor (Rapigest SF) b. MALDI sample prep target plate 3. Whole protein multi-dimensional LC/MS 4. Nanospray ESI LC/MS/MS of protein digests 1. Column technology 2. Multi-dimensional nanohplc 5. Interface for LC/MALDI (LC-MALDIprep)

3 Life Sciences: Analyzing the -omes omes Genome DNA instructions Transcriptome mrna work list Proteome proteins functionality Metabolome small molecules outcome

4 The primary justification for Genomic and Proteomic research is to improve the process of diagnosis and treatment of disease. protein identification protein expression post-translational modifications (PMTs) protein-protein interactions antagonist binding site

5 The Nature of the Problem Biological systems are complex and always changing Active biomolecules are often present in trace amounts Interactions between molecules are crucial Complete understanding of how and why things happen is years away

6 Common Themes Separations technology Mass spectrometry Automation Informatics Standardization Miniaturization

7 Analytical Workflow for Proteomics Complex Protein Mixtures from Cells, Tissues, BioFluids 2-D Gels 1-D Gels Digestion Digestion Digestion HPLC Spot on Plates nanolc-esi-ms/ms Deposit onto Plate MALDI-TOF MS Protein Databases MALDI-MS/MS

8 Proteins to Peptides to MS

9 MS Tools: MALDI Matrix-Assisted Laser Desorption Ionization 337 nm pulse from N 2 laser ions sample crystallized with UV absorbing matrix target plate

10 MS Tools: MALDI-TOF Peptide Mass Fingerprinting digestion protein(s) peptides 1) gi Score: 178 mw pi 6.75 hemoglobin, beta [Homo sapiens] sequence coverage: 43% MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLS TPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVD PENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH Protein ID % intensity database search MALDI-TOF MS m/z

11 MS Tools: Q-TOF Q LC/MS/MS

12 Data-Directed Directed Analysis (DDA) for LC/MS/MS of peptides with Q-TOFQ

13 Goals of the Waters Life Sciences Initiative To leverage the strengths of Waters and Micromass in becoming a leading supplier of consumables, new HPLC products, and mass spectrometry technology to address the Life Sciences market consumables HPLC MS

14 Proteomics Technologies Complex Protein Mixtures from Cells, Tissues, BioFluids Alliance 2796MD RapiGest TM SF 2-D Gels RapiGest TM SF Digestion Spot on Plates MALDI Target Plate MALDI-TOF MS Digestion 1-D Gels RapiGest TM SF Digestion nanolc columns nanolc-esi-ms/ms Modular CapLC Protein Databases HPLC Deposit onto Plate LC-MALDIprep MALDI-MS/MS

15 RapiGest TM SF A new acid-cleavable chemical reagent which aids digestion of proteins and is compatible with mass spectrometry

16 Trypsin Activity in the Presence Denaturing Reagents: RapiGest TM SF versus SDS Trypsin Activity A 252 nm No Surfactant 0.1% 0.5% 2.5% } 0.1% SDS 0.5% SDS RapiGest TM SF minutes

17 RapiGest TM SF Reduces Digestion Time Intact Myoglobin Digestion Time Course Monitored by LC/ESI-TOF MS (50 mm NH 4 CO 3 )

18 Compatibility of RapiGest TM SF with MS Analysis A) 100 % MALDI-TOF mass spectra of 500 fmol of BSA digest A) w/o RapiGest TM SF B) 0.25% RapiGest TM, followed by addtion of 50 mm HCl w/o RapiGest TM SF B) % 0.25% RapiGest TM SF m/z

19 Fast Protein Digestion: MALDI-TOF MS Peptide Mass Fingerprinting trypsin digestion in 50 mm NH 4 HCO 3 versus 0.1% RapiGest TM SF, digestion time = 5 minutes Proteins Myoglobin Amino Acid Sequence Coverage (%) Post column 50 mm NHconfiguration 4 HCO 3 0% 0.1% RapiGest TM SF 54% Bovine Ubiquitin Ovalbumin 0% 0% 100% 25% Bovine Serum Albumin 23% 33%

20 Solubilization and Digestion of Hydrophobic Proteins: Bacteriorhodopsin MALDI-TOF MS Spectra A)1 hour digestion with trypsin in 8 M urea and ZipTip TM clean up B) 1 hour digestion with trypsin in 0.25% RapiGest TM followed by addition of 50 mm HCl A) 8M Urea ZipTip TM B) 0.25% RapiGest TM SF w/o ZipTip TM * bacteriorhodopsin peptides * trypsin autolysis peptides m/z

21 RapiGest TM SF Summary Provides rapid digestion of proteins Acid-cleavable reagent which does not interfere with mass spectral analysis Eliminates Post the column need for high configuration concentrations of denaturing agents (8 M urea, SDS, etc.), which are difficult to completely remove and are detrimental to MS analysis A solubilization agent for hydrophobic proteins

22 MALDI Sample Preparation Target Plate Sample preparation with concentration/desalting directly on the MALDI target plate

23 MALDI Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry Current technology allows measurements on biopolymers (peptides, proteins, antibodies, DNA, enzymes) Easy to use Sensitive (subfemtomole detection of peptides) Peptide mass mapping (or fingerprinting ) for Protein Identification 1D gel spots 2D gel spots Major requirement: samples must be relatively clean Salts, buffers, detergents, etc. must be removed, or diluted out, otherwise ion signals of biomolecules are suppressed

24 Current Methods for High-Throughput MALDI Sample Preparation Millipore ZipTip TM Poor recovery ( < 50%) Poor recovery in presence of certain buffers and detergents (urea, SDS, etc.) Sample Post preconcentration column is configuration limited Mulitple sample manipulation steps through resin Bruker Anchorchip TM Preconcentrates only No clean up or desalting capabilities (efficient washing of samples on plate is limited)

25 Modified Sample Preparation Target Plate for MALDI PTFE film Sample Moat Adsorptive region (active well)

26 Sample Preparation Target Plate for MALDI Objectives Specifically designed for biopolymers Compatible with automated robotics (MassPrep TM ) Facilitates sample clean-up (desalting and surfactant removal) Concentrates (increase sensitivity) Minimize sample manipulation Suitable for on-plate chemistry (digest, derivatization) Useful for proteomics/biopolymer applications Superior to competitive technologies (Millipore ZipTip TM, Bruker AnchorChip TM )

27 Sample Prep Target Plate for MALDI Sample Concentration Sample is placed onto plate As sample dries, it is concentrated into the sample well Biopolymers will bind onto the well Salts and surfactants are easily washed away

28 Sample Concentration Effects of the New MALDI Target Plate Peptide Mixture: 1 fmol/µl

29 Sample Desalting Comparison: 1M Urea Removal of 1 M Urea from 100 fmol of standard peptide mixture Modified Target Plate, After wash with 0.1% TFA ZipTip C18 sample applied to stainless steel plate 100 fmol Peptide, No Urea

30 Detergent Removal Comparison: 0.1% SDS Removal of 0.1% SDS from 10 fmol of standard peptide mixture Modified Target Plate, After wash with 0.1% TFA ZipTip C18 sample applied to stainless steel plate 10 fmol Peptide, No 0.1% SDS

31 Analysis of Samples from In-gel Digestion with Modified MALDI Target Plate MALDI spectrum of 1µL of in-gel digest of Ovalbumin (~200 fmol) from silver-stained 1-D gel band, sample was washed three times with 0.1% TFA before the addition of matrix solution.

32 Modified MALDI Target Plate Summary Provides means for sample concentration and clean up directly on the target plate without sample loss High capacity: sample volumes up to 10 ul can be applied Sample can be washed to remove high concentrations of salts and detergents Significantly enhances the sensitivity of MALDI-TOF-MS with limits of detection in the sub-femtomole range Robust and simple to use Sample preparation with plate can be fully automated with robotic workstation (MassPrep TM System)

33 New HPLC System: Alliance 2796MD for Multidimensional Chromatography Parallel 2D Chromatography Prototype 2795 Symmetry 300 C4 (2.1 x 50 mm) Shodex SP (4.6 x 35 mm) Valve 1 Waste Symmetry 300 C4 (2.1 x 50 mm) MS UV Fraction Collector

34 Post Column Configuration Waste x 95 cm Port 7 of Valve 1 Model Valve x 20 cm x 30 cm UV-Vis Detector Split ratio 8:1 Fraction Collector II 50 um x 25 cm 60 um x 18.5 cm Union LCT Probe

35 Model Sample for 2D LC/MS Analysis: Yeast Ribosomal Proteins Crystal Structure of a Halobacterial Large Ribosomal Subunit Ban et al. (2000) Science 289:

36 Preparation of a Ribosomal Protein Fraction Yeast Cleared Lysate Ultracentrifugation Prep1 Prep 2, 3 Post column configuration 78 Subunits 116 Proteins 43 Subunits 64 Proteins

37 2D Chromatograms: Yeast Ribosomal Sample L1218A_23 Sm (SG, 1x3) Channel An1 UV SCX Gradient Steps 2.92e4 % L1218_ MS Time TOF MS ES+ TIC 2.59e4 % Time

38 OpenLynx TM Automated Data Processing for 2-D LC/ESI ESI-TOF MS Analysis: Fraction 3 OpenLynx TM Controlled Data Processing Peak ID Sum Spectra over Peak Deconvolute Spectra Generate Report Peak ApexTrack TM Combine Spectra MaxEnt1 TM Raw m/z Spectra Deconvoluted Spectra

39 Identification of Selected Ribosomal Protein Peaks from Fraction 3 Multiply charged MS spectrum Max Ent Deconvolution Protein MW Retention Time (min) Observed mass Assignment Calculated MW L26A-M L33A-M L33B-M L38-M L31A-M L31B-M L6A-M+Ac L6B-M S15-M+Ac S19A-M S9B-M L9A or or L9B-M+Ac L5-M

40 Results of Complete 2-D D (SCX/RP) LC/ESI ESI-TOF MS Analysis of Yeast Ribosomal Proteins AB AB AB AB 5 6 AB Small Subunit 33 / 47 Isoforms (70%) 27 / 32 Subunits (84%) AB 23 AB 24 AB AB 1 AB 2 AB Large Subunit 52 / 69 Isoforms (75%) 40 / 46 Subunits (87%) 8 15 AB AB 12 AB 19 AB AB AB Observed AB 37 P AB P1 P2 41 AB 42 AB Not Observed 85% of all known ribosomal subunit proteins identified 30 additional proteins identified Average mass accuracy of ppm achieved in replicate experiments

41 Alliance 2796MD LC/MS Summary Fully-automated, flexible multidimensional system for protein separation/fractionation Capable of resolving and isolating proteins which are difficult to analyze by traditional 2D gel electrophoresis (basic, acidic, hydrophobic, etc.) ESI-TOF LC/MS provides molecular weight information to identify proteins with ppm mass accuracy Data processing for obtaining protein molecular weight from LC/ESI-TOF MS raw data can be fully automated using OpenLynx TM Fractions of proteins can be collected and digested for further MS analysis (MALDI, nano-esi, LC/MS/MS)

42 LC/MS Consumables Nano-LC columns, trapping columns, 2-D chromatography of peptides

43 Goals of Nano-LC for Proteomics LC/MS/MS Applications Maximize peak capacity to resolve complex mixtures of peptides Achieve high sensitivity for MS and MS/MS detection Minimize sample losses Minimize dead volume Reduce background and chemical noise from solvent

44 Waters Symmetry PicoFrit TM and IntegraFrit TM Nano-LC/MS columns Symmetry C18 75 µm x 10 cm PicoFrit column Symmetry C18 75 µm x 10 cm IntegraFrit column

45 Interfacing MS to Nanoscale LC Columns Micromass PicoFrit TM Sprayer 75 micron id column packing HV Pico-frit column Frit from Stream Select module low volume union nebuliser gas inlet nanoflow x,y,z stage

46 CapLC TM and Q-TOF2Q TM

47 Waters Symmetry TM C18 PicoFrit TM Column LC/MS of 250 fmol of Apomyoglobin Digest 05-Mar :51:49 apomyo250fmol_test_ Sm (SG, 2x3) 100 peak widths <15 sec (FWHM) TOF MS ES+ TIC 2.98e % Time

48 Opti-Pak TM Format for Capillary-, Nano-LC fitting Directly connects to port of switching valve All hardware included 5 per pack column Symmetry C18, 5 µm, 0.3 x 5 mm trapping column PolyLC SCX 0.3 x 5 mm column

49 Goals of 2D Chromatography 1. Possible alternative/complementary strategy for 2D gel electrophoresis 2. Reduce complexity of protein digest mixtures with second dimension 3. Enhance sensitivity for peptides originating from difficult proteins : low copy number highly basic (high PI) highly acidic (low PI) membrane post-translationally modified

50 Schematic of Multi-Dimensional Nano-LC System on CapLC TM Injection Needle Autosampler Valve RP Desalting/ Trapping Column C SCX Waste 10 Port RPC Vent Valve UV and/or MS Autosampler Tray Solvent C A B Sample Vials Solvent D Vials Solvents A & B Solvent D: Step Elution Buffers of Increasing Ionic Strength

51 2-D D Chromatography Interfaced to Q-TOFQ Waters Opti-Pak TM SCX column 10-port valve Waters Opti-Pak TM Symmetry TM C18 trap column Sample cone Splitter tee Mixing tee PicoFrit TM Symmetry TM C18 75 µm x 10 cm nanolc column

52 2-D D NanoLC/MS/MS of Enriched Ribosomal Subunit Protein Digest BPI chromatograms of nine elution steps with KCl 2D_051502_9 1: TOF MS ES mm KCl BPI % D_051502_8 1: TOF MS ES mm KCl BPI % D_051502_7 1: TOF MS ES mm KCl BPI % D_051502_6 1: TOF MS ES BPI % D_051502_5 1: TOF MS ES mm KCl BPI % D_051502_4 1: TOF MS ES mm KCl BPI % D_051502_3 1: TOF MS ES mm KCl BPI % D_051502_2 1: TOF MS ES mm KCl BPI % D_051502_1 1: TOF MS ES mm KCl BPI % Time mm KCl

53 Data Directed Analysis Chromatograms of the Six Most Intense Precursor Ions (charge states +2, +3): 25 mm KCl Elution Step ~100 MS/MS spectra were acquired for the 25 mm KCl step. From these spectra, there were 39 hits in the database, with 23 yielding a score of > 11 6th most intense precursor ion 5th most intense precursor ion 4th most intense precursor ion 3rd most intense precursor ion 2nd most intense precursor ion Most intense precursor ion Survey Scan TIC 2D_051502_2 100 % 0 2D_051502_2 100 % 0 2D_051502_ % 0 2D_051502_2 100 % 7: TOF MSMS ES BPI : TOF MSMS ES BPI : TOF MSMS ES+ BPI 45 4: TOF MSMS ES+ BPI D_051502_2 3: TOF MSMS ES BPI % D_051502_2 2: TOF MSMS ES BPI % D_051502_2 1: TOF MS ES BPI % Time

54 MS/MS Spectrum of Ribosomal Tryptic Peptide from 25 mm KCl Elution Step Peptide Sequence: AVQDNGESAFR; Protein ID: Yeast riobsome subunit L13a m/z , charge: 2, mw: ; delta: 0.021; score: 14.3

55 Example Display of ProteinLynx TM Global Server Database Search Results for the 150 mm KCl Elution Step

56 Summary of Yeast Ribosomal Subunit Protein Identification from Database Search of MS/MS Data Acquired for each KCl Elution Step Subunit MW Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Step 9 (kd) L L L L L L L P P L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L P L L L L *Color scheme Scores Red 31+ Orange Yellow White 0-10 (No ID)

57 Results of 2-D 2 NanoLC/MS/MS Method A fully automated 2-D LC/MS/MS method at the nanolc scale has been demonstrated for a complex digest of yeast large ribosomal subunit proteins A total of 34 out of 46 (74%) possible proteins were identified The total analysis time of the 2-D method was ~12.5 hours Three-column configuration improves robustness, avoiding introduction of salts/buffers into the analytical nanolc column and MS source

58 Summary of Capillary-, NanoLC Consumables IntegraFrit TM and PicoFrit TM 75 µm Symmetry C18 columns provide high peak capacity and good resolution required for nanolc/ms/ms separations PicoFrit TM Symmetry C18 column allows direct nanospray introduction into the ion source, minimizing dead volume and potential sample loss Direct-connect OPTI-PAK TM columns low backpressure good recovery for subfemtomole levels of peptide format allows easy configuration and replacement of the SCX and trap/desalt columns with a 6-,10-port valve for either a 1-D or 2-D method Symmetry C18 OPTI-PAK TM trap column is robust and can effectively remove buffers and high concentrations of salts through many injection cycles

59 New Product: LC-MALDIprep Sample Deposition Module LC-MALDIprep Waters CapLC Interface between HPLC and MALDI

60 Description of the LC-MALDIprep The LC-MALDIprep is a sample deposition module that serves as an interface between reversed-phase HPLC and MALDI mass spectrometry Uses a heated capillary nebulizer to desolvate column eluent and spray it onto a MALDI target pre-coated with matrix Compatible RP HPLC flow rate range from 5 to 50 µl/min Computer control software sets the velocity of the sample stage, the temperature of the nozzle and the collection pattern The module can initiate a series of sample collections based on remote inject-start signals This device is compatible with a wide range of HPLC and MALDI manufacturers

61 LC-MALDIprep Utilizes an Adhesive Target which is Pre-coated with Matrix The pre-coated matrix targets are a one time use consumable item

62 Mounting the Target on the MALDI plate 1. Mounting 2. Assembled target plate 3. LC-MALDIprep Plate Holder LC-MALDIprep Target Assembly in Plate Holder With Lane Shield Mounted jig

63 The Heated Capillary Nebulizer LC Eluent N 2 Heated Nebulizer Gas Block Heater Metal Capillary Sample Spot and Stripe Collection Patterns Analyte Deposition X-Y Stage Motion MALDI Plate

64 LC-MALDIprep Software Deposition in Tracks sample deposition in tracks temperature gradient

65 LC-MALDIprep Software Deposition in Spots sample deposition in spots temperature gradient

66 The Base Peak Intensity Chromatogram from LC/MALDI-TOF/MS Analysis of an Apomyoglobin Tryptic Digest Separation 100 T T T T T % 9.12 T T Scan

67 Sample for Comparing LC-MALDIprep vs. ZipTip & Spotting Tryptic digest of high abundance protein in the presence of low abundance proteins Alcohol Dehdrogenase (1250 fmol) Actin (10 fmol) Myoglobin (10 fmol) Cytochrome c (10 fmol) Compare detection of tryptic peptides and protein sequence coverage of a capillary LC/MS MALDI separation vs. ZipTip TM & spotting

68 MALDI LC/MS of 4 Protein Tryptic Digest CapLC with Waters Symmetry C x 150 mm column H 2 O/Acetonitrile gradient with 0.1% TFA fmol ADH 10 fmol actin 10 fmol apomyoglobin 10 fmol cytochrome c BPI Time (minutes)

69 A LC-MALDI 100 Actin: T33-34 KYSVWIGGSILR % Comparison of Mass Spectral S/N for Tryptic Peptides from Actin (10 fmol) ) in the Four Protein Digest LC-MALDIprep versus ZipTip TM and Spot Actin: T23 SYELPDGQVITIGNER Actin: T28 DLYANNVMSGGTTMYPGIADR Post column % configuration % B Spot % % % m/z m/z m/z

70 Peptide Mass Mapping of a Four Protein Digest Mixture: LC-MALDI vs. ZipTip TM Number of Fits Number of Peptides Matched Actin ADH Myoglobin Protein Digest 10 fmol 1270 fmol 10 fmol LC-MALDI # peptides ZipTip # peptides CytC 10 fmol Percent Coverage Percent Protein Sequence Covered Actin 10 fmol LC-MALDI % Sequence ADH 10 fmol Myoglobin 10 fmol Protein Digest ZipTip % Sequence CytC 10 fmol

71 LC-MALDI QTOF Analysis of a Global Digest of Yeast Ribosomal Proteins Accession Score Match MW pi Description number (P10664) S RIBOSOMAL PROTEIN L4-A (L2A) (RP2) (P14126) S RIBOSOMAL PROTEIN L3 (YL1) (RP1) (TR Query m/z Charge Delta Score YAQDGAGIER Accession number Score Match MW pi Description (P14126) S RIBOSOMAL PROTEIN L3 (YL1) (RP1) (TR Analyze complex digest Collect separation to 94 fractions Consume target protein only

72 LC-MALDIprep Summary Collection of analyte from wide range of RP HPLC separation flow rates: capillary and micro bore (5 to 50 ul/min) Robust LC-MALDI leads to reduced ion suppression effects and interference between peptides of similar m/z Improved peptide mass fingerprinting versus ZipTip TM and spot method Improved MALDI MS/MS (QTOF or TOF/TOF) peptide sequencing results due to reduction in sample complexity Stable, long-term storage of collected separations that can be analyzed months later Work is in progress for interfacing to nanolc columns

73 The Future: Building the Product Matrix for Life Sciences Equipment?? Mass Spec Sample Prep Post column HPLC configuration??? HPLC columns? Analysis?? Consumables

74 Life Sciences Applied Technology Bob Pfeifer Steven Cohen Robert Plumb Scott Berger Bob Karol Daniel Wall Chris Stumpf Jennifer Granger Hongi Liu Asish Chakroborty Acknowledgements Life Sciences Chemistry John Gebler Martin Gilar Peter Lee Weiben Chen Ying-Qing Yu Amy Daly Ken Fountain Advance Separations Bruce Compton Ed Bouvier Geoff Gerhardt Micromass UK Jim Langridge Jeff Brown Dominic Gostick Emanuelle Claude Allan Milar Chemistry Operations Dorothy Phillips Bruce Smith Mike Savaria

75 Thank you!

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