Mass spectrometry based proteomics

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Mass spectrometry based proteomics"

Transcription

1 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Mass spectrometry based proteomics Zsuzsanna Darula Institute of Biochemistry November 25, 2015

2 Proteomics: large scale study of proteins Proteome: the complete set of proteins expressed by a cell / tissue / organism at a given time under defined conditions

3 Separation / fractionation of protein samples 1.: gel electrophoresis 1D: SDS PAGE (size separation, limited resolution, low complexity samples) 2D: IEF SDS PAGE isoelectric point size resolving power: a few thousand proteins/gel limitations: membrane proteins highly acidic / basic proteins very small/ large proteins 16 BAC/SDS PAGE size separation in both dimensions, limited resolution Native gel SDS PAGE analysis of protein complexes: isolation of complex in 1 st dimension, separation of components in the second

4 Separation / fractionation of protein samples 2.: Chromatography Type Abbr. Principle of separation Size exclusion chromatography SEC Differences in size and shape Ion exchange chromatography IEC Electrostatic interactions (pk a, pk b ) Normal phase chromatography Hydrophilic interaction chromatography Reversed phase chromatography Hydrophobic interaction chromatography NPC HILIC RPC HIC Polar interactions Dispersive interactions Affinity chromatography AC Specific interactions

5 Identification of separated proteins 1. Edman sequencing REAGENT REAGENT H 2 N Amino Acid 1 AA 2 AA 3 AA 4 AA 5 Amino Acid 1 AA 2 AA 3 AA 4 AA 5 N reagent Phenylisothiocyanate (PITC) C S H 2 N R HN O Peptide ph:8 first amino acid unit H, heat Amino Acid 1 REAGENT H 2 N AA 2 AA 3 AA 4 AA 5 S N NH R H 2 N Peptide HPLC O Phenylthiohydantoin amino acid reagent product requires pure protein requires free protein N terminus (acetylation?) ~30 (max 50 60) AA can be determined sample requirements: 10 pmol slow (~ 1h / AA) ~ isobaricaa s can be distinguished (I/L; Q/K)

6 Identification of separated proteins 2. Western blot sensitive specific (semi)quantitative requires antibody foreknowledge of protein of interest is required expensive

7 Identification of separated proteins 3. Mass Spectrometry (MS) Sensitive (fmol/amol range) Quick No antibody or external standards required Amenable with mixtures blocked peptides modified peptides (PTM analysis) Problems with distinguishing isobaric AA s (Leu/Ile (and Gln/Lys))

8 Mass Spectrometry (MS) Determination of m/z value of gas phase ions (mass to charge ration, m: mass, z: charge) VACUUM SYSTEM spectrum sample ION SOURCE MASS ANALYZER DETECTOR signal Int. Formation of ions Separation of ions Detection of ions m/z

9 Ion source generation of gas phase ions soft ionization techniques MALDI: matrix assisted laser desorption ionization, singly charged ions ESI: electrospray ionization, multiply charged ions Mass analyzer separation of ions according to m/z defines performance of the mass spectrometer Sensitivity Resolution Mass accuracy (absolute / relative (ppm!!!)) Linear dynamic range Speed Mass range analyzers used in proteomics: Quadrupole, Ion trap, flight tube (TOF), FT ICR, Orbitrap (alone or in combo)

10 Theorethical peak shape of peptide m/z: 1000 (z=1) at different resolution R=500 R=1000 R=2000 R=10000

11 Monoisotopic mass only 12 C, 1 H, 14 N, 16 O (and 32 S) x 13 C(and ) Element Mass number Natural occurrence % 2500 C x 13 C(and ) H O N S

12 Monoisotopic peak is ALWAYS the first in the isotope cluster but not necessarily the most abundant! m/z: 300 m/z: 1800 m/z: 3000 m/z: 5000

13 Isotope spacing enables charge determination _48_6PM # 1907 RT: AV: 1 NL: 1.67E5 T: FTMS p NSI Full ms [ ] Relative Abundance Δ m/z: 1 z= Δ m/z: 1/2 z= Δ m/z: 1/3 z= m/z

14 MS analysis of proteins Top down Intact proteins are investigated Bottom up Peptides generated from proteins are investigated Peptide mass fingerprinting (For low complexity samples) Tandem mass spectrometry (For high(er) complexity samples)

15 TOP DOWN APPROACH molecular weight determination of intact proteins fragmentation of intact isolated proteins (ETD, ECD, CID, HCD) degradation products sequence variants combinations of post translational modifications positions of disulfide bridges techniqually challenging lower MW proteins limited sample complexity high resolution (R> !), special instrument required

16 Bottom up approach Generation of peptides from proteins Enzymatically Endopeptidase Specificity ph range Trypsin R, K Chymotrypsin Y, F, W, L Glu C E, D Asp N D Arg C R Lys C K Lys N N Chemically Cyanogen bromide (Met (Trp )) Acidic hydrolysis (Asp, (Glu ))

17 Enzymatic protocols most often use trypsin Cleaves after Lys and Arg (except the next AA is a Pro) Provides at least one basic amino acid per peptide (facilitates ion generation for MS) Statistically the size of the tryptic peptides are perfect for m/z range of analyzers (~10% of the AA content is R or K) Cheap, reliable, known sequence Modified trypsin is available commercially ( autolysis)

18 Peptide mass fingerprinting (PMF) 2D gel electrophoresis reduction alkylation trypsin peptide extraction desalting MALDI TOF Database Search Int. Protein list m/z MS compatible staining Reduction: DTT (dithiothreitol), mercaptoethanol, TCEP (tris(2 carboxyethyl)phosphine) Alkylation: IAM (iodoacetamide), IAA (iodoacetic acid), NEM (N Ethylmaleimide), 4 vinylpyridine

19 MALDI Ion Source (Matrix Assisted Laser Desorption Ionization) Laser beam hν Target Plate Desorption Desolvation Ionization to Analyzer Co crystallized matrix and analyte molecules Protein or peptide analyte Acidic matrix molecule O Commonly used matrices O O HO OH OH H 3 CO OH OH HO CN HO OCH 3 DHB CHCA SA 2,5 DihydroxyBenzoic Acid α Cyano 4 HydroxyCinnamic Acid Sinapinic Acid

20 TOF Analyzer (time of flight) Repeller Laser beam Grid ( pole) Detector Spectrum Signal Int. Plate m/z Analytes with different m/z values (different velocity different amount of time to reach the detector) generated ions are accelerated by applied electric field ions of same charge have the same kinetic energy velocity of the ions depends on their mass to charge ratio time to reach the detector is measured, m/z calculated 2 2 t V m z 2 or t m z L

21 MALDI TOF spectrum of tryptic digest of a ~70 kda protein a.i m /z

22 Search engines: Mascot, Protein Prospector

23

24

25

26

27

28 100 % sequence coverage? peptide m/z out of detection range (short and long peptides) poor recovery of hydrophobic peptides (extraction from gel) loss of hydrophilic peptides (desalting) ion suppression (MS analysis) incorrect sequence in database protein processing (signal peptide, propeptide)

29 MALDI-TOF MS, without fractionation 3 different components T T

30 PMF SUMMARY Protein is cleaved into peptides in a specific manner (usually using trypsin) The m/z value of the resulting peptides is determined using mass spectrometry (typically MALDI TOF) Protein is identified by database search by comparing experimentally determined peptide m/z s to theoretical ones generated in silico from proteins present in database Applicable to low complexity samples (a few protein/sample)

31 For more complex peptide mixtures: MS/MS based protein ID Ion source ionization - Analyzer 1 ionization selection fragmentation ionization Analyzer 2 m/z separation Detector detection sample Int. Tandem in space (two separate analyzers, Q TOF, Q TRAP, IT Orbitrap ) Tandem in time (single analyzer, ion traps) m/z MS/MS (fragmentation) spectrum

32 Electrospray ionization (ESI) (heated) capillary e - to the analyzer - High voltage Sample nebulized to fine aerosol Size of sample droplets is reduced by applied electric field and heat (desolvation) Droplets explode when electrostatic repulsion overcomes surface tension Finally completely desolved ions enter the mass analyzer Multiply charged ions are formed On line coupling to HPLC: LC MS/MS

33 FRAGMENTATION Energy based Energy is put into the peptide (weakest bonds break) Collision Induced Dissociation (CID/CAD, HCD) Infra Red MultiPhoton Dissociation (IRMPD) Radical based Electrons create unstable radical ions that spontaneously fragment at sites of electron capture Electron Capture Dissociation (ECD) Electron Transfer Dissociation (ETD)

34 Sequence ions are formed by fragmentation of the peptide backbone: SPECIFIC TO PEPTIDE SEQUENCE! x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 H 2 N R 1 O R 2 N H H N O R 3 O R 4 N H O OH a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3

35 Collision induced dissociation (CID) 1. Sequence ions H 2 S A M P L E R OH y 6 y 5 y 4 y 3 y 2 y 1 b 1 b 2 b 3 b 4 b 5 b 6 b fragment ions y fragment ions 6 H S A M P L E R H 2 OH 1 5 H S A M P L E R H 2 OH 2 4 H S A M P H 2 L E R OH H H S A M S A H 2 P L E R OH H M P L E R OH H S H A M P L E R OH 2 6

36 Instrument dependent fragmentation Fragment ions generated by CID: 1. Sequence ions (a, b, y) 2. Internal fragments (if multiple fragmentation) 3. Satellite ions (water and NH 3 loss of fragment ions) 4. Immonium ions (info on amino acid content)

37 LC MS/MS: data dependent analysis RT: Relative Abundance BPI Time (min) NL: 2.72E7 Base Peak F: ms MS _27 Ions eluting from HPLC _27 #8826 RT: AV: 1 NL: 4.20E6 T: FTMS p NSI Full ms [ ] z=2 100 Relative Abundance MS z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= m/z z=2 Ions detected in Orbitrap analyzer at 41.43min _27 #8827 RT: AV: 1 NL: 2.90E5 T: ITMS c NSI d w Full ms2 [ ] Relative Abundance MS / MS m/z MS/MS spectrum of precursor ion m/z: (2), fragmented and measured in ion trap

38 1. Generation of peak list (txt, mgf, dta ): List of all MS/MS data acquired during an LC MS/MS experiment: For each MS/MS spectra: precursor m/z, precursor charge state list of m/z and intensity values for observed fragment ions 2. Database search All MS/MS spectra is searched individually, results are given as proteins listing peptides assigned to them Search engine (Mascot, Protein Prospector, OMSSA, Sequest, Proteome Discoverer, Byonic ) 1. defines peptide candidates with the same (theoretical) m/z that is observed for the precursor (in silico digestion of proteins in the database) 2. compares observed fragment ion list to theoretical fragment ion list of peptide candidates (instrument dependent fragmentation!!!) 3. assigns a score and / or a probability value (expectation value, E value) to peptide matches for deciding the goodness of identifications 4. lists the highest scored match(es) for each peptide ID meeting acceptance criteria

39

40

41

42

43

44 One peptide to multiple proteins? Homology Between species Protein families within the same species Different isoforms of the same protein Coincidence

45 Sample Gene Acc.No. Protein Protein MW Unique Peptides Coverage 1 At3g02090 Q42290 mitochondrial-processing peptidase % beta subunit, (MPP beta) 1 At3g16480 O04308 mitochondrial-processing peptidase 54 1 a 3.00% alpha subunit 2, (MPP alpha-2) 1 At2g07727 P42792 Cytochrome b (MTCYB) (COB) b 3.30% (CYTB) 2 At1g51980 Q9ZU25 mitochondrial-processing peptidase 54 c % alpha subunit 1 (MPP Alpha-1) 2 At3g16480 O04308 mitochondrial-processing peptidase 54 c 1 c (9) 16.40% alpha subunit 2, (MPP alpha-2) 3 At5g40810 Q0WNJ4 Cytochrome c1 (CYC1-2) % 4 At5g13430 Q9LYR3 Ubiquinol--cytochrome-c reductase (REISKE subunit) 5 At4g32470 Q9SUU5 Ubiquinol-cytochrome C reductase complex 14 kda protein 5 At5g25450 Q3E953 Ubiquinol-cytochrome C reductase complex 14 kda protein % 14.5 d % 14.6 d 1 d (2) 18.00%

46

47

48 All MS/MS data assigned to peptides? NO non peptide components (salts, detergents, derivatizing agents ) incomplete reduction / alkylation upon sample preparation post translational modifications side reactions during sample preparation cyclization (N terminal Gln, Cys(CAM)) methylation (Glu, Asp) oxidation (Met, Trp, Cys, Tyr) carbamylation (N term, Lys) carbamidomethylation (N term, Lys, Met, His, Asp) deamidation (Asn, Gln) S acrylamide formation (Cys) formation of alkali metal adducts of peptides nonspecific cleavages incomplete digestion (number of missed cleavages?) multiple peptides selected and fragmented in source fragmentation resulting in nonspecific peptides in source water loss (Ser, Thr, Asp, Glu) short peptides may not yield enough fragments for confident ID low quality spectra incorrect monoisotopic m/z incorrect charge state peptides/proteins not present in database

49 Post translational modifications 1. proteolytic cleavages N terminal Met cleavage signal peptide propeptide chemical group acetylation, phosphorylation, glycosylation, ubiquitination, sumoylation, lipidation... intra or inter peptidic linkages disulfide bonds...

50 Post translational modifications 2. (if) reflected in the molecular weight of the protein and corresponding peptide: amenable to MS usually substoichiometric: requires enrichment of modified peptides prior to MS modified peptides may feature similar or different fragmentation pattern: alternative fragmentation techniques (e.g. ETD for glycopeptides) characteristic fragmentation: pinpointing modified peptides (carbohydrate oxonium ions for glycopeptides, neutral loss ions for phosphopeptides / Met oxidized peptides)

51 Phosphopeptides fragment similarly to unmodified peptides (CID) Characteristic 98 Da neutral loss (phosphate) HVG _szbk_01 57 (21.531) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (57:58) : TOF MSMS ES 7.71e phosphopeptide peptide % 0 % _szbk_02 5 (21.980) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (5:6) Da Da 98 Da Da Da Da 80 Da : TOF MSMS ES m/z

52 Glycopeptides show different fragmentation than unmodified peptides (CID) y Relative Abundance y TPIVGQPSIPGGPVR b 6 b b y y 8 * b (2) y 5 y y 10 8 y m/z y _02 #2601 RT: AV: 1 NL: 1.53E4 T: ITMS p NSI t d Full ms2 [ ] Relative Abundance Sialic acid related oxonium ions Carbohydrate loss related fragment ions Same peptide glycosylated (HexNAcHexSA) m/z

53 Quantitative proteomics 1. Gel based 2D gel electrophoresis time consuming, labor intensive limited dynamic range not suitable for LMW ( 15kDa) and HMW (>150 kda) protein hydrophobic (e.g. membrane) proteins insoluble proteins needs min. 100 g total protein/gel 3 replicates / sample Improvements more sensitive staining large format higher resolving gel sample prefractionation DIGE (Difference Gel Electrophoresis, fluorescent labeling)

54 Quantitative proteomics 2. Gel free MS based Stable isotopic labeling ( 2 H, 13 C, 15 N, 18 O) Chemical labeling ICAT (isotope coded affinity tag, Cys) itraq, TMT (Multiplexed isobaric tagging technology, Lys/peptide N term) ICPL (isotope coded protein label) Formaldehyde NaBH 3 CN (peptide N term, Lys) Enzymatic labeling 16 O/ 18 O exchange catalyzed by trypsin (peptide C term) Metabolic labeling SILAC (Stable isotope labeling with amino acids in cell culture) 15 N or 13 C labeling (complete or partial) Label free LC MS quantification comparison of ion signal intensities (spectral counting), or chromatographic peak areas (MS E )

55 Cell/ tissue SILAC ICAT N/C term. itraq, TMT Internal Standards Protein Peptide MS

56 Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project.

Proteomics in Practice

Proteomics in Practice Reiner Westermeier, Torn Naven Hans-Rudolf Höpker Proteomics in Practice A Guide to Successful Experimental Design 2008 Wiley-VCH Verlag- Weinheim 978-3-527-31941-1 Preface Foreword XI XIII Abbreviations,

More information

PROTEIN SEQUENCING. First Sequence

PROTEIN SEQUENCING. First Sequence PROTEIN SEQUENCING First Sequence The first protein sequencing was achieved by Frederic Sanger in 1953. He determined the amino acid sequence of bovine insulin Sanger was awarded the Nobel Prize in 1958

More information

Aiping Lu. Key Laboratory of System Biology Chinese Academic Society APLV@sibs.ac.cn

Aiping Lu. Key Laboratory of System Biology Chinese Academic Society APLV@sibs.ac.cn Aiping Lu Key Laboratory of System Biology Chinese Academic Society APLV@sibs.ac.cn Proteome and Proteomics PROTEin complement expressed by genome Marc Wilkins Electrophoresis. 1995. 16(7):1090-4. proteomics

More information

Introduction to Proteomics 1.0

Introduction to Proteomics 1.0 Introduction to Proteomics 1.0 CMSP Workshop Tim Griffin Associate Professor, BMBB Faculty Director, CMSP Objectives Why are we here? For participants: Learn basics of MS-based proteomics Learn what s

More information

LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program

LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program

More information

ProteinScape. Innovation with Integrity. Proteomics Data Analysis & Management. Mass Spectrometry

ProteinScape. Innovation with Integrity. Proteomics Data Analysis & Management. Mass Spectrometry ProteinScape Proteomics Data Analysis & Management Innovation with Integrity Mass Spectrometry ProteinScape a Virtual Environment for Successful Proteomics To overcome the growing complexity of proteomics

More information

Session 1. Course Presentation: Mass spectrometry-based proteomics for molecular and cellular biologists

Session 1. Course Presentation: Mass spectrometry-based proteomics for molecular and cellular biologists Program Overview Session 1. Course Presentation: Mass spectrometry-based proteomics for molecular and cellular biologists Session 2. Principles of Mass Spectrometry Session 3. Mass spectrometry based proteomics

More information

Mass Spectrometry Based Proteomics

Mass Spectrometry Based Proteomics Mass Spectrometry Based Proteomics Proteomics Shared Research Oregon Health & Science University Portland, Oregon This document is designed to give a brief overview of Mass Spectrometry Based Proteomics

More information

Tutorial for Proteomics Data Submission. Katalin F. Medzihradszky Robert J. Chalkley UCSF

Tutorial for Proteomics Data Submission. Katalin F. Medzihradszky Robert J. Chalkley UCSF Tutorial for Proteomics Data Submission Katalin F. Medzihradszky Robert J. Chalkley UCSF Why Have Guidelines? Large-scale proteomics studies create huge amounts of data. It is impossible/impractical to

More information

Introduction to Proteomics

Introduction to Proteomics Introduction to Proteomics Åsa Wheelock, Ph.D. Division of Respiratory Medicine & Karolinska Biomics Center asa.wheelock@ki.se In: Systems Biology and the Omics Cascade, Karolinska Institutet, June 9-13,

More information

AB SCIEX TOF/TOF 4800 PLUS SYSTEM. Cost effective flexibility for your core needs

AB SCIEX TOF/TOF 4800 PLUS SYSTEM. Cost effective flexibility for your core needs AB SCIEX TOF/TOF 4800 PLUS SYSTEM Cost effective flexibility for your core needs AB SCIEX TOF/TOF 4800 PLUS SYSTEM It s just what you expect from the industry leader. The AB SCIEX 4800 Plus MALDI TOF/TOF

More information

In-Depth Qualitative Analysis of Complex Proteomic Samples Using High Quality MS/MS at Fast Acquisition Rates

In-Depth Qualitative Analysis of Complex Proteomic Samples Using High Quality MS/MS at Fast Acquisition Rates In-Depth Qualitative Analysis of Complex Proteomic Samples Using High Quality MS/MS at Fast Acquisition Rates Using the Explore Workflow on the AB SCIEX TripleTOF 5600 System A major challenge in proteomics

More information

Challenges in Computational Analysis of Mass Spectrometry Data for Proteomics

Challenges in Computational Analysis of Mass Spectrometry Data for Proteomics Ma B. Challenges in computational analysis of mass spectrometry data for proteomics. SCIENCE AND TECHNOLOGY 25(1): 1 Jan. 2010 JOURNAL OF COMPUTER Challenges in Computational Analysis of Mass Spectrometry

More information

Master course KEMM03 Principles of Mass Spectrometric Protein Characterization. Exam

Master course KEMM03 Principles of Mass Spectrometric Protein Characterization. Exam Exam Master course KEMM03 Principles of Mass Spectrometric Protein Characterization 2010-10-29 kl 08.15-13.00 Use a new paper for answering each question! Write your name on each paper! Aids: Mini calculator,

More information

Laboration 1. Identifiering av proteiner med Mass Spektrometri. Klinisk Kemisk Diagnostik

Laboration 1. Identifiering av proteiner med Mass Spektrometri. Klinisk Kemisk Diagnostik Laboration 1 Identifiering av proteiner med Mass Spektrometri Klinisk Kemisk Diagnostik Sven Kjellström 2014 kjellstrom.sven@gmail.com 0702-935060 Laboration 1 Klinisk Kemisk Diagnostik Identifiering av

More information

Analysis of proteins

Analysis of proteins Analysis of proteins Western blot Protein seperation (liqiuid chromatography) Mass spectrometry Assaying of protein in... Blood (e.g. viral infections, pregnancy test) Cells Tissue Urin (bladder infection)

More information

--not necessarily a protein! (all proteins are polypeptides, but the converse is not true)

--not necessarily a protein! (all proteins are polypeptides, but the converse is not true) 00Note Set 5b 1 PEPTIDE BONDS AND POLYPEPTIDES OLIGOPEPTIDE: --chain containing only a few amino acids (see tetrapaptide, Fig 5.9) POLYPEPTIDE CHAINS: --many amino acids joined together --not necessarily

More information

Quan%ta%ve proteomics. Maarten Altelaar, 2014

Quan%ta%ve proteomics. Maarten Altelaar, 2014 Quan%ta%ve proteomics Maarten Altelaar, 2014 Proteomics Altelaar et al. Nat Rev Gen 14, 2013, 35-48 Quan%ta%ve proteomics Quan%ta%ve proteomics Control Diseased, s%mulated, Knock down, etc. How quan%ta%ve

More information

Methods for Protein Analysis

Methods for Protein Analysis Methods for Protein Analysis 1. Protein Separation Methods The following is a quick review of some common methods used for protein separation: SDS-PAGE (SDS-polyacrylamide gel electrophoresis) separates

More information

A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys

A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys Questions- Proteins & Enzymes A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys Reaction of the intact peptide

More information

Electrospray Ion Trap Mass Spectrometry. Introduction

Electrospray Ion Trap Mass Spectrometry. Introduction Electrospray Ion Source Electrospray Ion Trap Mass Spectrometry Introduction The key to using MS for solutions is the ability to transfer your analytes into the vacuum of the mass spectrometer as ionic

More information

Advantages of the LTQ Orbitrap for Protein Identification in Complex Digests

Advantages of the LTQ Orbitrap for Protein Identification in Complex Digests Application Note: 386 Advantages of the LTQ Orbitrap for Protein Identification in Complex Digests Rosa Viner, Terry Zhang, Scott Peterman, and Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA,

More information

Mass Spectrometry for Chemists and Biochemists

Mass Spectrometry for Chemists and Biochemists Erasmus Intensive Program SYNAPS Univ. of Crete - Summer 2007 Mass Spectrometry for Chemists and Biochemists Spiros A. Pergantis Assistant Professor of Analytical Chemistry Department of Chemistry University

More information

using ms based proteomics

using ms based proteomics quantification using ms based proteomics lennart martens Computational Omics and Systems Biology Group Department of Medical Protein Research, VIB Department of Biochemistry, Ghent University Ghent, Belgium

More information

Principles and Applications of Proteomics

Principles and Applications of Proteomics Principles and Applications of Proteomics Why Proteomics? 2-DE Overview Sample preparation 1 st & 2 nd dimension seperation Data Analysis Sample preparation for Mass Spectrometry Mass Spectrometry MALDI-TOF,

More information

Introduction to Proteomics

Introduction to Proteomics Introduction to Proteomics Why Proteomics? Same Genome Different Proteome Black Swallowtail - larvae and butterfly Biological Complexity Yeast - a simple proteome 6,113 proteins = 344,855 tryptic peptides

More information

Introduction to Mass Spectrometry. W.M. Keck Biomedical Mass Spectrometry Lab

Introduction to Mass Spectrometry. W.M. Keck Biomedical Mass Spectrometry Lab Introduction to Mass Spectrometry W.M. Keck Biomedical Mass Spectrometry Lab Moore Health Sciences Library Rooms 1335 & 1337 May 18, 2010 The Keck Mass Spectrometry Lab of the Biomolecular Resource Facility

More information

Introduction to mass spectrometry (MS) based proteomics and metabolomics

Introduction to mass spectrometry (MS) based proteomics and metabolomics Introduction to mass spectrometry (MS) based proteomics and metabolomics Tianwei Yu Department of Biostatistics and Bioinformatics Rollins School of Public Health Emory University September 10, 2015 Background

More information

Choices, choices, choices... Which sequence database? Which modifications? What mass tolerance?

Choices, choices, choices... Which sequence database? Which modifications? What mass tolerance? Optimization 1 Choices, choices, choices... Which sequence database? Which modifications? What mass tolerance? Where to begin? 2 Sequence Databases Swiss-prot MSDB, NCBI nr dbest Species specific ORFS

More information

Thermo Scientific PepFinder Software A New Paradigm for Peptide Mapping

Thermo Scientific PepFinder Software A New Paradigm for Peptide Mapping Thermo Scientific PepFinder Software A New Paradigm for Peptide Mapping For Conclusive Characterization of Biologics Deep Protein Characterization Is Crucial Pharmaceuticals have historically been small

More information

La Protéomique : Etat de l art et perspectives

La Protéomique : Etat de l art et perspectives La Protéomique : Etat de l art et perspectives Odile Schiltz Institut de Pharmacologie et de Biologie Structurale CNRS, Université de Toulouse, Odile.Schiltz@ipbs.fr Protéomique et Spectrométrie de Masse

More information

Application Note # LCMS-81 Introducing New Proteomics Acquisiton Strategies with the compact Towards the Universal Proteomics Acquisition Method

Application Note # LCMS-81 Introducing New Proteomics Acquisiton Strategies with the compact Towards the Universal Proteomics Acquisition Method Application Note # LCMS-81 Introducing New Proteomics Acquisiton Strategies with the compact Towards the Universal Proteomics Acquisition Method Introduction During the last decade, the complexity of samples

More information

Global and Discovery Proteomics Lecture Agenda

Global and Discovery Proteomics Lecture Agenda Global and Discovery Proteomics Christine A. Jelinek, Ph.D. Johns Hopkins University School of Medicine Department of Pharmacology and Molecular Sciences Middle Atlantic Mass Spectrometry Laboratory Global

More information

Chapter 3. Protein Structure and Function

Chapter 3. Protein Structure and Function Chapter 3 Protein Structure and Function Broad functional classes So Proteins have structure and function... Fine! -Why do we care to know more???? Understanding functional architechture gives us POWER

More information

Protein Purification and Analysis

Protein Purification and Analysis Protein Purification and Analysis Numbers of genes: Humans ~40,000 genes Yeast ~6000 genes Bacteria ~3000 genes Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Some proteins

More information

Definition of the Measurand: CRP

Definition of the Measurand: CRP A Reference Measurement System for C-reactive Protein David M. Bunk, Ph.D. Chemical Science and Technology Laboratory National Institute of Standards and Technology Definition of the Measurand: Human C-reactive

More information

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras Molecular Cell Biology Prof. D. Karunagaran Department of Biotechnology Indian Institute of Technology Madras Module 5 Methods in Cell Biology (Methods to Manipulate Protein, DNA and RNA and Methods to

More information

Labeling Technologies for Quantitative Protein Expression Analysis

Labeling Technologies for Quantitative Protein Expression Analysis Innovative Solutions for Quantitative Protein and Biomarker Research Labeling Technologies for Quantitative Protein Expression Analysis Understanding Disease States via Quantitative Protein Expression

More information

Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit

Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit Fast, simple and cost effective analysis Many areas of biochemical research and

More information

泛 用 蛋 白 質 體 學 之 質 譜 儀 資 料 分 析 平 台 的 建 立 與 應 用 Universal Mass Spectrometry Data Analysis Platform for Quantitative and Qualitative Proteomics

泛 用 蛋 白 質 體 學 之 質 譜 儀 資 料 分 析 平 台 的 建 立 與 應 用 Universal Mass Spectrometry Data Analysis Platform for Quantitative and Qualitative Proteomics 泛 用 蛋 白 質 體 學 之 質 譜 儀 資 料 分 析 平 台 的 建 立 與 應 用 Universal Mass Spectrometry Data Analysis Platform for Quantitative and Qualitative Proteomics 2014 Training Course Wei-Hung Chang ( 張 瑋 宏 ) ABRC, Academia

More information

Ionization of amino acids

Ionization of amino acids Amino Acids 20 common amino acids there are others found naturally but much less frequently Common structure for amino acid COOH, -NH 2, H and R functional groups all attached to the a carbon Ionization

More information

(c) How would your answers to problem (a) change if the molecular weight of the protein was 100,000 Dalton?

(c) How would your answers to problem (a) change if the molecular weight of the protein was 100,000 Dalton? Problem 1. (12 points total, 4 points each) The molecular weight of an unspecified protein, at physiological conditions, is 70,000 Dalton, as determined by sedimentation equilibrium measurements and by

More information

Proteome Discoverer 1.3 Software: Enhanced Tools For Protein Identification

Proteome Discoverer 1.3 Software: Enhanced Tools For Protein Identification Proteome Discoverer 1.3 Software: Enhanced Tools For Protein Identification David Horn Proteomics Software Strategic Marketing Manager September 22, 2011 Hybrid Orbitrap Mass Spectrometers CID HCD ETD

More information

MASCOT Search Results Interpretation

MASCOT Search Results Interpretation The Mascot protein identification program (Matrix Science, Ltd.) uses statistical methods to assess the validity of a match. MS/MS data is not ideal. That is, there are unassignable peaks (noise) and usually

More information

Quantitative mass spectrometry in proteomics: a critical review

Quantitative mass spectrometry in proteomics: a critical review Anal Bioanal Chem (2007) 389:1017 1031 DOI 10.1007/s00216-007-1486-6 REVIEW Quantitative mass spectrometry in proteomics: a critical review Marcus Bantscheff & Markus Schirle & Gavain Sweetman & Jens Rick

More information

Workshop IIc. Manual interpretation of MS/MS spectra. Ebbing de Jong. Center for Mass Spectrometry and Proteomics Phone (612)625-2280 (612)625-2279

Workshop IIc. Manual interpretation of MS/MS spectra. Ebbing de Jong. Center for Mass Spectrometry and Proteomics Phone (612)625-2280 (612)625-2279 Workshop IIc Manual interpretation of MS/MS spectra Ebbing de Jong Why MS/MS spectra? The information contained in an MS spectrum (m/z, isotope spacing and therefore z ) is not enough to tell us the amino

More information

Effects of Intelligent Data Acquisition and Fast Laser Speed on Analysis of Complex Protein Digests

Effects of Intelligent Data Acquisition and Fast Laser Speed on Analysis of Complex Protein Digests Effects of Intelligent Data Acquisition and Fast Laser Speed on Analysis of Complex Protein Digests AB SCIEX TOF/TOF 5800 System with DynamicExit Algorithm and ProteinPilot Software for Robust Protein

More information

Pesticide Analysis by Mass Spectrometry

Pesticide Analysis by Mass Spectrometry Pesticide Analysis by Mass Spectrometry Purpose: The purpose of this assignment is to introduce concepts of mass spectrometry (MS) as they pertain to the qualitative and quantitative analysis of organochlorine

More information

SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data

SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data Automated Data Interpretation for Glycan Characterization Jenny Albanese 1, Matthias Glueckmann 2 and Christof Lenz 2 1 AB

More information

Retrospective Analysis of a Host Cell Protein Perfect Storm: Identifying Immunogenic Proteins and Fixing the Problem

Retrospective Analysis of a Host Cell Protein Perfect Storm: Identifying Immunogenic Proteins and Fixing the Problem Retrospective Analysis of a Host Cell Protein Perfect Storm: Identifying Immunogenic Proteins and Fixing the Problem Kevin Van Cott, Associate Professor Dept. of Chemical and Biomolecular Engineering Nebraska

More information

Error Tolerant Searching of Uninterpreted MS/MS Data

Error Tolerant Searching of Uninterpreted MS/MS Data Error Tolerant Searching of Uninterpreted MS/MS Data 1 In any search of a large LC-MS/MS dataset 2 There are always a number of spectra which get poor scores, or even no match at all. 3 Sometimes, this

More information

ProteinPilot Report for ProteinPilot Software

ProteinPilot Report for ProteinPilot Software ProteinPilot Report for ProteinPilot Software Detailed Analysis of Protein Identification / Quantitation Results Automatically Sean L Seymour, Christie Hunter SCIEX, USA Pow erful mass spectrometers like

More information

Lecture 15: Enzymes & Kinetics Mechanisms

Lecture 15: Enzymes & Kinetics Mechanisms ROLE OF THE TRANSITION STATE Lecture 15: Enzymes & Kinetics Mechanisms Consider the reaction: H-O-H + Cl - H-O δ- H Cl δ- HO - + H-Cl Reactants Transition state Products Margaret A. Daugherty Fall 2004

More information

Mass Spectrometry Signal Calibration for Protein Quantitation

Mass Spectrometry Signal Calibration for Protein Quantitation Cambridge Isotope Laboratories, Inc. www.isotope.com Proteomics Mass Spectrometry Signal Calibration for Protein Quantitation Michael J. MacCoss, PhD Associate Professor of Genome Sciences University of

More information

Principles of Mass Spectrometry-Based Proteomics. Steven Gygi Department of Cell Biology Harvard Medical School, Boston, MA

Principles of Mass Spectrometry-Based Proteomics. Steven Gygi Department of Cell Biology Harvard Medical School, Boston, MA Principles of Mass Spectrometry-Based Proteomics Steven Gygi Department of Cell Biology Harvard Medical School, Boston, MA The Flow of Information The Flow of Information Networks Complexes Proteins Genes

More information

Experimental workflow

Experimental workflow Experimental workflow Pg. 3 Lysis Protein quant Protein precipitation Pg. 4 Digest Pgs. 5&6 Label Peptides Peptide quant Ratio check Search, filter & compile data Pgs. 9-12 Combine Samples LC-MS3 Pg. 8

More information

Building innovative drug discovery alliances. Evotec Munich. Quantitative Proteomics to Support the Discovery & Development of Targeted Drugs

Building innovative drug discovery alliances. Evotec Munich. Quantitative Proteomics to Support the Discovery & Development of Targeted Drugs Building innovative drug discovery alliances Evotec Munich Quantitative Proteomics to Support the Discovery & Development of Targeted Drugs Evotec AG, Evotec Munich, June 2013 About Evotec Munich A leader

More information

Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush

Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush α-keratins, bundles of α- helices Contain polypeptide chains organized approximately parallel along a single axis: Consist

More information

ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRY

ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRY Annu. Rev. Biochem. 2001. 70:437 73 Copyright c 2001 by Annual Reviews. All rights reserved ANALYSIS OF PROTEINS AND PROTEOMES BY MASS SPECTROMETRY Matthias Mann, 1,2 Ronald C. Hendrickson, 2 and Akhilesh

More information

Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008

Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008 Bruker Daltonics Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008 In the ABRF-PRG study 2008 [*] the ability to characterize

More information

Mass spectrometry. Dr. Kevin R. Tucker

Mass spectrometry. Dr. Kevin R. Tucker Mass spectrometry Dr. Kevin R. Tucker What is mass spectrometry? A versatile analytical technique used to determine the composition of chemical samples either at the atomic or molecular level. Ionic forms

More information

Pep-Miner: A Novel Technology for Mass Spectrometry-Based Proteomics

Pep-Miner: A Novel Technology for Mass Spectrometry-Based Proteomics Pep-Miner: A Novel Technology for Mass Spectrometry-Based Proteomics Ilan Beer Haifa Research Lab Dec 10, 2002 Pep-Miner s Location in the Life Sciences World The post-genome era - the age of proteome

More information

The Scheduled MRM Algorithm Enables Intelligent Use of Retention Time During Multiple Reaction Monitoring

The Scheduled MRM Algorithm Enables Intelligent Use of Retention Time During Multiple Reaction Monitoring The Scheduled MRM Algorithm Enables Intelligent Use of Retention Time During Multiple Reaction Monitoring Delivering up to 2500 MRM Transitions per LC Run Christie Hunter 1, Brigitte Simons 2 1 AB SCIEX,

More information

Quantitative mass spec based proteomics

Quantitative mass spec based proteomics Quantitative mass spec based proteomics Tuula Nyman Institute of Biotechnology tuula.nyman@helsinki.fi Proteomics is the large-scale study of proteins Proteomics provides information on: -protein expression

More information

The Organic Chemistry of Amino Acids, Peptides, and Proteins

The Organic Chemistry of Amino Acids, Peptides, and Proteins Essential rganic Chemistry Chapter 16 The rganic Chemistry of Amino Acids, Peptides, and Proteins Amino Acids a-amino carboxylic acids. The building blocks from which proteins are made. H 2 N C 2 H Note:

More information

TECHNICAL BULLETIN. ProteoMass Peptide & Protein MALDI-MS Calibration Kit. Catalog Number MSCAL1 Store at Room Temperature

TECHNICAL BULLETIN. ProteoMass Peptide & Protein MALDI-MS Calibration Kit. Catalog Number MSCAL1 Store at Room Temperature ProteoMass Peptide & Protein MALDI-MS Calibration Kit Catalog Number MSCAL1 Store at Room Temperature TECHNICAL BULLETIN Description This kit provides a range of standard peptides and proteins for the

More information

Overview. Triple quadrupole (MS/MS) systems provide in comparison to single quadrupole (MS) systems: Introduction

Overview. Triple quadrupole (MS/MS) systems provide in comparison to single quadrupole (MS) systems: Introduction Advantages of Using Triple Quadrupole over Single Quadrupole Mass Spectrometry to Quantify and Identify the Presence of Pesticides in Water and Soil Samples André Schreiber AB SCIEX Concord, Ontario (Canada)

More information

Biochemistry 2000 Sample Question Proteins. (1) Identify the secondary structure described in each of the following statements:

Biochemistry 2000 Sample Question Proteins. (1) Identify the secondary structure described in each of the following statements: (1) Identify the secondary structure described in each of the following statements: a. A coiled peptide chain held in place by hydrogen bonding between peptide bonds in the same chain b. A structure that

More information

Statistics and Algorithms for Peptide Mass Fingerprinting

Statistics and Algorithms for Peptide Mass Fingerprinting Statistics and Algorithms for Peptide Mass Fingerprinting Dipl.-Math. Hans-Michael Kaltenbach Thesis submitted to the Faculty of Technology, Bielefeld University, Germany for the degree of Dr. rer. nat.

More information

NUVISAN Pharma Services

NUVISAN Pharma Services NUVISAN Pharma Services CESI MS Now available! 1st CRO in Europe! At the highest levels of quality. LABORATORY SERVICES Equipment update STATE OF THE ART AT NUVISAN CESI MS Now available! 1st CRO in Europe!

More information

Absolute quantification of low abundance proteins by shotgun proteomics

Absolute quantification of low abundance proteins by shotgun proteomics Absolute quantification of low abundance proteins by shotgun proteomics Dr. Stefanie Wienkoop www.proteomefactory.com In cooperation with: Max-Planck-Institut für Molekulare Pflanzenphysiologie Stable

More information

Agilent G2721AA/G2733AA Spectrum Mill MS Proteomics Workbench

Agilent G2721AA/G2733AA Spectrum Mill MS Proteomics Workbench Agilent G2721AA/G2733AA Spectrum Mill MS Proteomics Workbench Application Guide Agilent Technologies Notices Agilent Technologies, Inc. 2012 No part of this manual may be reproduced in any form or by any

More information

Increasing the Multiplexing of High Resolution Targeted Peptide Quantification Assays

Increasing the Multiplexing of High Resolution Targeted Peptide Quantification Assays Increasing the Multiplexing of High Resolution Targeted Peptide Quantification Assays Scheduled MRM HR Workflow on the TripleTOF Systems Jenny Albanese, Christie Hunter AB SCIEX, USA Targeted quantitative

More information

Protein Physics. A. V. Finkelstein & O. B. Ptitsyn LECTURE 1

Protein Physics. A. V. Finkelstein & O. B. Ptitsyn LECTURE 1 Protein Physics A. V. Finkelstein & O. B. Ptitsyn LECTURE 1 PROTEINS Functions in a Cell MOLECULAR MACHINES BUILDING BLOCKS of a CELL ARMS of a CELL ENZYMES - enzymatic catalysis of biochemical reactions

More information

Quantitative proteomics background

Quantitative proteomics background Proteomics data analysis seminar Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals post transcriptional regulation of key cellular processes de Groot, M., Daran

More information

ProSightPC 3.0 Quick Start Guide

ProSightPC 3.0 Quick Start Guide ProSightPC 3.0 Quick Start Guide The Thermo ProSightPC 3.0 application is the only proteomics software suite that effectively supports high-mass-accuracy MS/MS experiments performed on LTQ FT and LTQ Orbitrap

More information

Peptide bonds: resonance structure. Properties of proteins: Peptide bonds and side chains. Dihedral angles. Peptide bond. Protein physics, Lecture 5

Peptide bonds: resonance structure. Properties of proteins: Peptide bonds and side chains. Dihedral angles. Peptide bond. Protein physics, Lecture 5 Protein physics, Lecture 5 Peptide bonds: resonance structure Properties of proteins: Peptide bonds and side chains Proteins are linear polymers However, the peptide binds and side chains restrict conformational

More information

SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays

SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays Protocol SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays Contact us: InfoLine: +49-30-6392-7878 Order per fax: +49-30-6392-7888 or e-mail: www: peptide@jpt.com www.jpt.com

More information

MRMPilot Software: Accelerating MRM Assay Development for Targeted Quantitative Proteomics

MRMPilot Software: Accelerating MRM Assay Development for Targeted Quantitative Proteomics MRMPilot Software: Accelerating MRM Assay Development for Targeted Quantitative Proteomics With Unique QTRAP and TripleTOF 5600 System Technology Targeted peptide quantification is a rapidly growing application

More information

Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following?

Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following? MCAT Question Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following? A. Carboxylic group and amino group B. Two carboxylic

More information

Week 9: MS in Space and Proteomics

Week 9: MS in Space and Proteomics Week 9: MS in Space and Proteomics 1 Last Time Detectors Small Molecule Applications, Environmental: (e.g. TWQC) 2 Mass Spectrometry in Space Possibly the coolest application of small molecule MS is in

More information

for mass spectrometry calibration tools Thermo Scientific Pierce Controls and Standards for Mass Spectrometry

for mass spectrometry calibration tools Thermo Scientific Pierce Controls and Standards for Mass Spectrometry Thermo Scientific Pierce Controls and Standards for Mass Spectrometry calibration tools for mass spectrometry Ensure confidence in instrument performance with Thermo Scientific Pierce Calibration Solutions

More information

Proteomic data analysis for Orbitrap datasets using Resources available at MSI. September 28 th 2011 Pratik Jagtap

Proteomic data analysis for Orbitrap datasets using Resources available at MSI. September 28 th 2011 Pratik Jagtap Proteomic data analysis for Orbitrap datasets using Resources available at MSI. September 28 th 2011 Pratik Jagtap The Minnesota http://www.mass.msi.umn.edu/ Proteomics workflow Trypsin Protein Peptides

More information

Biochemistry - I. Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II

Biochemistry - I. Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II Biochemistry - I Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II In the last class we studied the enzyme mechanisms of ribonuclease A

More information

IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon. V. Polypeptides and Proteins

IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon. V. Polypeptides and Proteins IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon A. Acid/Base properties 1. carboxyl group is proton donor! weak acid 2. amino group is proton acceptor! weak base 3. At physiological ph: H

More information

Analytical Testing Methods

Analytical Testing Methods Analytical Testing Methods Updated: February 2005 Background The Mandatory Guidelines for Federal Workplace Drug Testing Programs require a laboratory to conduct two analytical tests before a urine specimen

More information

Top five list for Mass Spectrometry. 1. Molecular weight 2. Fragmentation pattern 3. Isotope ratio 4. Nitrogen rule 5. Exact mass

Top five list for Mass Spectrometry. 1. Molecular weight 2. Fragmentation pattern 3. Isotope ratio 4. Nitrogen rule 5. Exact mass Mass Spectrometry Top five list for Mass Spectrometry 1. Molecular weight 2. Fragmentation pattern 3. Isotope ratio 4. Nitrogen rule 5. Exact mass A Mass Spectrometer A mass spectrometer is designed to

More information

In-depth Analysis of Tandem Mass Spectrometry Data from Disparate Instrument Types* S

In-depth Analysis of Tandem Mass Spectrometry Data from Disparate Instrument Types* S Research In-depth Analysis of Tandem Mass Spectrometry Data from Disparate Instrument Types* S Robert J. Chalkley, Peter R. Baker, Katalin F. Medzihradszky, Aenoch J. Lynn, and A. L. Burlingame Mass spectrometric

More information

AMD Analysis & Technology AG

AMD Analysis & Technology AG AMD Analysis & Technology AG Application Note 120419 Author: Karl-Heinz Maurer APCI-MS Trace Analysis of volatile organic compounds in ambient air A) Introduction Trace analysis of volatile organic compounds

More information

An algorithm for isoelectric point estimation

An algorithm for isoelectric point estimation An algorithm for isoelectric point estimation David L. Tabb Created 7/10/01 Updated 6/28/03 1 Introduction One of the most common techniques for separating mixtures of proteins is the two-dimensional polyacrylamide

More information

Proteomics Workflows & Technologies

Proteomics Workflows & Technologies Proteomics Workflows & Technologies Proteomics "The analysis of the entire PROTEin complement expressed by a genome, or by a cell or tissue type." Wasinger VC et al, Electrophoresis 16 (1995) Proteomics

More information

SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data

SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data SimGlycan Software*: A New Predictive Carbohydrate Analysis Tool for MS/MS Data Automated Data Interpretation for Glycan Characterization Jenny Albanese1, Matthias Glueckmann2 and Christof Lenz2 1Applied

More information

Introduction to Database Searching using MASCOT

Introduction to Database Searching using MASCOT Introduction to Database Searching using MASCOT 1 Three ways to use mass spectrometry data for protein identification 1.Peptide Mass Fingerprint A set of peptide molecular masses from an enzyme digest

More information

STANFORD UNIVERSITY MASS SPECTROMETRY 333 CAMPUS DR., MUDD 175 STANFORD, CA 94305-5080

STANFORD UNIVERSITY MASS SPECTROMETRY 333 CAMPUS DR., MUDD 175 STANFORD, CA 94305-5080 Training on the ZQ Open access MS Questions? Contact Dr. Allis Chien allis@stanford.edu 650-723-0710 0710 STANFORD UNIVERSITY MASS SPECTROMETRY STANFORD UNIVERSITY MASS SPECTROMETRY 333 CAMPUS DR., MUDD

More information

Chapter 14. Modeling Experimental Design for Proteomics. Jan Eriksson and David Fenyö. Abstract. 1. Introduction

Chapter 14. Modeling Experimental Design for Proteomics. Jan Eriksson and David Fenyö. Abstract. 1. Introduction Chapter Modeling Experimental Design for Proteomics Jan Eriksson and David Fenyö Abstract The complexity of proteomes makes good experimental design essential for their successful investigation. Here,

More information

THE ABC S (AND XYZ S) OF PEPTIDE SEQUENCING

THE ABC S (AND XYZ S) OF PEPTIDE SEQUENCING TE ABC S (AD XYZ S) F PEPTIDE SEQUECIG anno Steen* and Matthias Mann Abstract Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the

More information

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep Guide to Reverse Phase SpinColumns Chromatography for Sample Prep www.harvardapparatus.com Contents Introduction...2-3 Modes of Separation...4-6 Spin Column Efficiency...7-8 Fast Protein Analysis...9 Specifications...10

More information

Amino Acids, Peptides, Proteins

Amino Acids, Peptides, Proteins Amino Acids, Peptides, Proteins Functions of proteins: Enzymes Transport and Storage Motion, muscle contraction Hormones Mechanical support Immune protection (Antibodies) Generate and transmit nerve impulses

More information

Interpretation of MS-Based Proteomics Data

Interpretation of MS-Based Proteomics Data Interpretation of MS-Based Proteomics Data Yet-Ran Chen, 陳 逸 然 Agricultural Biotechnology Research Center Academia Sinica Brief Overview of Protein Identification Workflow Protein Sample Specific Protein

More information

Investigating Biological Variation of Liver Enzymes in Human Hepatocytes

Investigating Biological Variation of Liver Enzymes in Human Hepatocytes Investigating Biological Variation of Liver Enzymes in Human Hepatocytes MS/MS ALL with SWATH Acquisition on the TripleTOF Systems Xu Wang 1, Hui Zhang 2, Christie Hunter 1 1 AB SCIEX, USA, 2 Pfizer, USA

More information