Tandem mass spectra were extracted from the Xcalibur data system format. (.RAW) and charge state assignment was performed using in house software
|
|
- June Richard
- 5 years ago
- Views:
Transcription
1 Supplementary Methods Software Interpretation of Tandem mass spectra Tandem mass spectra were extracted from the Xcalibur data system format (.RAW) and charge state assignment was performed using in house software (RAW_Xtractor). Tandem mass spectra were interpreted by SEQUEST 1, which was parallelized on a Beowulf cluster of ~35 computers 2 and results were filtered, sorted, and displayed using the DTASelect program. 3 Searches were performed against the combined human, mouse, rat, and yeast databases from RefSeq and the wormpep115 database from Extraction of Chromatograms After filtering the results from SEQUEST using DTASelect (+1 > 1.5, +2 > 2.1, +3 > 3.1, CN > 0.08), ion chromatograms were generated from either MS or tandem mass spectra using a modified version of a program previously written in our lab (RelEx). 4 This software is available from the authors for individual use and evaluation through an Institutional Software Transfer Agreement (see for details). In all cases, the Xcalibur.RAW files were used to generate chromatograms from the m/z range surrounding both the unlabeled and labeled precursor or fragment ions. The elemental compositions and corresponding isotopic distributions for both the unlabeled and labeled peptides and all potential +1 fragment ions were calculated. This information was then used to determine the appropriate m/z range from which to extract ion
2 intensities, which included all isotopes with greater than 10% of the calculated monoisotopic peak abundance. For extraction from tandem mass spectra, the peptide precursor m/z was used to identify the proper mass window containing the appropriate tandem mass spectrum for both the unlabeled and labeled peptides. Often, the fragmentation patterns for these peptides were in different windows, but in some instances (i.e., depending on the mass shift, charge state, and size of mass window employed) both unlabeled and labeled fragmentation patterns appeared in the same tandem mass spectrum. Regardless, the ion intensities from all predicted +1 fragment ions were summed and reconstructed ion chromatograms were stored in tab delimited text files with a.chro extension. Calculation of Peptide and Protein Ratios RelEx was used to calculate peptide ion intensity ratios for each pair of extracted ion chromatograms (i.e., for each.chro file). This program has already been described in detail 4, so it will only briefly be discussed here. The heart of the program is a linear least squares correlation that is used to calculate the ratio (i.e., slope of the line) and closeness of fit (i.e., correlation coefficient (r)) between the data points of the unlabeled and labeled ion chromatograms. Measured peptide ratios were then sorted by protein locus and outliers, as determined by both a Dixon s Q-test and Chauvenet s criteria 5, were thrown out. Protein ratios were then calculated from the mean of remaining peptide ratios. Chromatogram Filtering Poor quality chromatograms were filtered using a multi-step filtering process implemented in RelEx. First, chromatograms were required to have a sufficient signal to noise to accurately quantify the mixtures analyzed by requiring the larger peaks (i.e.,
3 light for our standard mixtures) have a signal to noise of at least 3.5 for the 1:1 mixture, and 7.5:1 for the 10:1 mixture. For the C.elegans samples, we required the larger peak (i.e., light or heavy ) have a signal to noise of at least 5:1. Next, we required chromatographic pairs to have a correlation coefficient between unlabeled and labeled signals of at least Finally, we used the statistical methods (i.e., Q-test, and Chauvenet s criteria) already described above to remove outliers when calculating protein ratios from the average of all peptide ratios. Cross correlation algorithm for calculation of peptide molecular weight from tandem mass spectra We have developed an approach to identify the molecular weight of a peptide ion directly from its corresponding tandem mass spectrum using a cross correlation function. In general, this technique can be successfully applied to a variety of tandem mass spectra; but, tandem mass spectra with charge states > +2, poor signal to noise, or unusual fragmentation are less successful. We have shown that the molecular weight can be calculated within ±3 amu for approximately 70-85% of tandem mass spectra obtained from a trypsin digested yeast whole cell lysate. The algorithm has also shown potential for use as a spectral quality filter. This strategy can be used to identify the precursor ion for tandem mass spectra acquired using large ion selection windows in Data-Independent Collision Induced Dissociation (DICID). In essence, a mass spectrum is compared to a reversed copy of the same spectrum using a cross correlation algorithm. In this manner, the correlation score typically maximizes when complementary b and y ions overlap. Because complementary b and y ions are formed from the cleavage of the same amide bond, their summation is equal to
4 the molecular weight of the peptide + 1. By calculating the offset between complementary b and y ion pairs, it is therefore, possible to determine the precursor mass. This technique has been successfully applied to a variety of spectra of different charge states and varying overall quality and typically results in a precursor mass calculation within 3 of the true value. Methods Preparation of C.elegans Samples Eggs were prepared by bleaching ( % NaOCl, 0.75N KOH) gravid adult worms at the 91 th hour. Both samples were washed thoroughly with the standard M9 buffer and resuspended in the lysis buffer (20 mm HEPES ph 7.4, 10 mm KCl, 1.5 mm MgCl 2, 1 mm DTT, 1x EDTA free protease inhibitor cocktail (Roche)). Eggs or young adults were lysed on ice in a Dounce homogenizer (KONTES 20) with 100 strokes and centrifuged at 1000g for 10 min at 4 C. The 14 N Eggs and 15 N young adults were mixed at a 1:1 protein ratio and subjected to a 100,000g spin at 4 C for 1 h to yield a soluble fraction and a membrane fraction. The soluble fraction was then subjected to methanol/chloroform precipitation, and the precipitated protein was resuspended in 100mM Tris ph 8.5, 8 M urea. Protein Digestion Before digestion, protein mixtures were denatured with solid urea (final concentration of 8M in 100mM Tris ph 8.5), reduced with TCEP (Sigma, 3 mm final concentration for 20 min. at room temp.), and alkylated with iodoacetamide (Sigma, 10 mm final concentration for 30 min at room temp.). Digestion was then performed using
5 a previously described protocol. 6 The resulting peptide mixture was acidified with formic acid (3%). MudPIT This approach has been described in detail by several authors 6-9, so it will only briefly be detailed here. A three phase microcapillary column was constructed and equilibrated with 5% acetonitrile / 0.1% formic acid for ~30 min before the peptide mixture was loaded. For analysis, the microcolumn was positioned in-line with a Surveyor autosampler and Surveyor quaternary HPLC pump (ThermoElectron) directly in front of the heated capillary opening of either an LCQ-Deca or LTQ ion trap mass spectrometer (ThermoElectron). Peptide mixtures were loaded on-line using the autosampler and analyzed using a six or twelve step separation procedure. 6, 8, 10 As peptides were eluted and ionized into the mass spectrometer, acquisition of tandem mass spectra was repeated continuously during the course of the analysis. All tandem mass spectra were collected using a normalized collision energy of 35% and only 1 µscan was acquired (i.e., no signal averaging was employed). Typical instrumental parameters included a maximum injection time of 100 ms, an MS/MS AGC target of 10,000 ions, and a spray voltage of 2.4kV. Scan Sequences and Search Parameters Qualitative Analysis: When data-dependent acquisition was employed, a cycle consisting of one full scan mass spectrum ( m/z) followed by three tandem mass spectra was repeated throughout the analysis, and an isolation window of 3 m/z was used. The dataindependent scheme employed consisted of 100 MS/MS scans interrogating the mass
6 range between m/z (i.e., each window had an isolation width of 10 m/z) and the scan cycle time was ~35s. Each sample was separated by a 4hr reverse phase gradient and analyzed on an LTQ mass spectrometer. Acquired tandem mass spectra were then searched against the combined human, mouse, rat, and yeast databases from Refseq using SEQUEST (peptide mass tolerance = 10, enzyme specificity = trypsin), and filtered using default DTASelect criteria (i.e., +1 > 1.5, +2 > 2.1, +3 > 3.1, CN > 0.08, min. # peptides = 2, tryptic status = half). Quantitative Analysis of Yeast Lysates: The data-independent acquisition scheme consisted of 1 MS scan followed by 20 MS/MS scans interrogating the mass range between m/z (an isolation width of 10 m/z). Each scan cycle was completed in ~4s. Acquired tandem mass spectra were then searched against the yeast-orfs database from Refseq. The MS scan was acquired entirely for chromatogram comparison purposes and no data-dependent acquisition was employed. Quantitative Analysis of C.elegans: The data-independent acquisition scheme consisted of 34 MS/MS scans interrogating the mass range between m/z (an isolation width of 15 m/z). Each scan cycle was completed in ~10s. Acquired tandem mass spectra were then searched against the wormpep115 database from using SEQUEST (mass accuracy = 15 amu, enzyme specificity = trypsin). Search results were then filtered with default DTASelect parameters (i.e., +1 > 1.8, +2 > 2.5, +3 > 3.5, CN > 0.08, and at least two peptides per locus).
7 Western Blots The PNS fractions of 14 N Eggs and 15 N young adults were prepared as above, centrifuged at 100,000g for 1 h at 4 C, and the supernatants were collected for Western blotting. A total of 10 µg proteins from 14 N Eggs or 15 N young adults were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for SQV-4, CRT-1, and CYP-5. The immunoreactive bands were visualized with SuperSignal, an enhanced chemiluminescent substrate (Pierce).
8 References 1. Eng, J.K., McCormack, A.L. & Yates, J.R., III An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 5, (1994). 2. Sadygov, R.G. et al. Code developments to improve the efficiency of automated MS/MS spectra interpretation. Journal of Proteome Research 1, (2002). 3. Tabb, D.L., McDonald, W.H. & Yates, J.R., III DTASelect and Contrast: Tools for Assembling and Comparing Protein Identifications from Shotgun Proteomics. Journal of Proteome Research 1, (2002). 4. MacCoss, M.J., Wu, C.C. & III, J.R.Y. A correlation algorithm for the automated analysis of quantitative shotgun proteomics data. Analytical Chemistry submitted (2003). 5. Taylor, J.R. An introduction to error analysis: the study of uncertainties in physical measurements. (University Science Books, Mill Valley, CA; 1982). 6. McDonald, W.H., Ohi, R., Miyamoto, D.T., Mitchison, T.J. & Yates, J.R. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. International Journal of Mass Spectrometry 219, (2002). 7. Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology 17, (1999).
9 8. MacCoss, M.J. et al. Shotgun identification of protein modifications from protein complexes and lens tissue. Proceedings of the National Academy of Sciences of the United States of America 99, (2002). 9. MacCoss, M.J., Wu, C.C. & Yates, J.R., III Probability-Based Validation of Protein Identifications Using a Modified SEQUEST Algorithm. Analytical Chemistry 74, (2002). 10. Washburn, M.P., Wolters, D. & Yates, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology 19, (2001).
Quantitation of a target protein in crude samples using targeted peptide quantification by Mass Spectrometry
Quantitation of a target protein in crude samples using targeted peptide quantification by Mass Spectrometry Jon Hao, Rong Ye, and Mason Tao Poochon Scientific, Frederick, Maryland 21701 Abstract Background:
More informationImproved 6- Plex TMT Quantification Throughput Using a Linear Ion Trap HCD MS 3 Scan Jane M. Liu, 1,2 * Michael J. Sweredoski, 2 Sonja Hess 2 *
Improved 6- Plex TMT Quantification Throughput Using a Linear Ion Trap HCD MS 3 Scan Jane M. Liu, 1,2 * Michael J. Sweredoski, 2 Sonja Hess 2 * 1 Department of Chemistry, Pomona College, Claremont, California
More informationHigh-Throughput Protein Quantitation Using Multiple Reaction Monitoring
High-Throughput Protein Quantitation Using Multiple Reaction Monitoring Application Note Authors Ning Tang, Christine Miller, Joe Roark, Norton Kitagawa and Keith Waddell Agilent Technologies, Inc. Santa
More informationMS-based proteomics to investigate proteins and their modifications
MS-based proteomics to investigate proteins and their modifications Francis Impens VIB Proteomics Core October th 217 Overview Mass spectrometry-based proteomics: general workflow Identification of protein
More informationTutorial 1: Setting up your Skyline document
Tutorial 1: Setting up your Skyline document Caution! For using Skyline the number formats of your computer have to be set to English (United States). Open the Control Panel Clock, Language, and Region
More informationProtein Quantitation II: Multiple Reaction Monitoring. Kelly Ruggles New York University
Protein Quantitation II: Multiple Reaction Monitoring Kelly Ruggles kelly@fenyolab.org New York University Traditional Affinity-based proteomics Use antibodies to quantify proteins Western Blot RPPA Immunohistochemistry
More informationAnalysis of Peptide MS/MS Spectra from Large-Scale Proteomics Experiments Using Spectrum Libraries
Anal. Chem. 2006, 78, 5678-5684 Analysis of Peptide MS/MS Spectra from Large-Scale Proteomics Experiments Using Spectrum Libraries Barbara E. Frewen, Gennifer E. Merrihew, Christine C. Wu, William Stafford
More informationWADA Technical Document TD2003IDCR
IDENTIFICATION CRITERIA FOR QUALITATIVE ASSAYS INCORPORATING CHROMATOGRAPHY AND MASS SPECTROMETRY The appropriate analytical characteristics must be documented for a particular assay. The Laboratory must
More informationProtein Quantitation II: Multiple Reaction Monitoring. Kelly Ruggles New York University
Protein Quantitation II: Multiple Reaction Monitoring Kelly Ruggles kelly@fenyolab.org New York University Traditional Affinity-based proteomics Use antibodies to quantify proteins Western Blot Immunohistochemistry
More informationMethods for proteome analysis of obesity (Adipose tissue)
Methods for proteome analysis of obesity (Adipose tissue) I. Sample preparation and liquid chromatography-tandem mass spectrometric analysis Instruments, softwares, and materials AB SCIEX Triple TOF 5600
More informationIsotopic-Labeling and Mass Spectrometry-Based Quantitative Proteomics
Isotopic-Labeling and Mass Spectrometry-Based Quantitative Proteomics Xiao-jun Li, Ph.D. Current address: Homestead Clinical Day 4 October 19, 2006 Protein Quantification LC-MS/MS Data XLink mzxml file
More informationFigure S1. Interaction of PcTS with αsyn. (a) 1 H- 15 N HSQC NMR spectra of 100 µm αsyn in the absence (0:1, black) and increasing equivalent
Figure S1. Interaction of PcTS with αsyn. (a) 1 H- 15 N HSQC NMR spectra of 100 µm αsyn in the absence (0:1, black) and increasing equivalent concentrations of PcTS (100 µm, blue; 500 µm, green; 1.5 mm,
More informationQuantitative Comparison of Proteomic Data Quality between a 2D and 3D Quadrupole Ion Trap
Quantitative Comparison of Proteomic Data Quality between a 2D and 3D Quadrupole Ion Trap Adele R. Blackler, Aaron A. Klammer, Michael J. MacCoss, and Christine C. Wu*, Department of Pharmacology, University
More informationModeling Mass Spectrometry-Based Protein Analysis
Chapter 8 Jan Eriksson and David Fenyö Abstract The success of mass spectrometry based proteomics depends on efficient methods for data analysis. These methods require a detailed understanding of the information
More informationNature Methods: doi: /nmeth Supplementary Figure 1. Fragment indexing allows efficient spectra similarity comparisons.
Supplementary Figure 1 Fragment indexing allows efficient spectra similarity comparisons. The cost and efficiency of spectra similarity calculations can be approximated by the number of fragment comparisons
More informationPeptideProphet: Validation of Peptide Assignments to MS/MS Spectra. Andrew Keller
PeptideProphet: Validation of Peptide Assignments to MS/MS Spectra Andrew Keller Outline Need to validate peptide assignments to MS/MS spectra Statistical approach to validation Running PeptideProphet
More informationDesigned for Accuracy. Innovation with Integrity. High resolution quantitative proteomics LC-MS
Designed for Accuracy High resolution quantitative proteomics Innovation with Integrity LC-MS Setting New Standards in Accuracy The development of mass spectrometry based proteomics approaches has dramatically
More informationComputational Methods for Mass Spectrometry Proteomics
Computational Methods for Mass Spectrometry Proteomics Eidhammer, Ingvar ISBN-13: 9780470512975 Table of Contents Preface. Acknowledgements. 1 Protein, Proteome, and Proteomics. 1.1 Primary goals for studying
More informationSelf-assembling covalent organic frameworks functionalized. magnetic graphene hydrophilic biocomposite as an ultrasensitive
Electronic Supplementary Material (ESI) for Nanoscale. This journal is The Royal Society of Chemistry 2017 Electronic Supporting Information for: Self-assembling covalent organic frameworks functionalized
More informationReagents. Affinity Tag (Biotin) Acid Cleavage Site. Figure 1. Cleavable ICAT Reagent Structure.
DATA SHEET Protein Expression Analysis Reagents Background The ultimate goal of proteomics is to identify and quantify proteins that are relevant to a given biological state; and to unearth networks of
More informationPurdue-UAB Botanicals Center for Age- Related Disease
Purdue-UAB Botanicals Center for Age- Related Disease MALDI-TOF Mass Spectrometry Fingerprinting Technique Landon Wilson MALDI-TOF mass spectrometry is an advanced technique for rapid protein identification
More informationThe Pitfalls of Peaklist Generation Software Performance on Database Searches
Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, June 1-5, 2008 The Pitfalls of Peaklist Generation Software Performance on Database Searches Aenoch J. Lynn,
More informationSpectrum-to-Spectrum Searching Using a. Proteome-wide Spectral Library
MCP Papers in Press. Published on April 30, 2011 as Manuscript M111.007666 Spectrum-to-Spectrum Searching Using a Proteome-wide Spectral Library Chia-Yu Yen, Stephane Houel, Natalie G. Ahn, and William
More informationSRM assay generation and data analysis in Skyline
in Skyline Preparation 1. Download the example data from www.srmcourse.ch/eupa.html (3 raw files, 1 csv file, 1 sptxt file). 2. The number formats of your computer have to be set to English (United States).
More informationMassHunter Software Overview
MassHunter Software Overview 1 Qualitative Analysis Workflows Workflows in Qualitative Analysis allow the user to only see and work with the areas and dialog boxes they need for their specific tasks A
More informationWADA Technical Document TD2015IDCR
MINIMUM CRITERIA FOR CHROMATOGRAPHIC-MASS SPECTROMETRIC CONFIRMATION OF THE IDENTITY OF ANALYTES FOR DOPING CONTROL PURPOSES. The ability of a method to identify an analyte is a function of the entire
More informationAccurate, High-Throughput Protein Identification Using the Q TRAP LC/MS/MS System and Pro ID Software
www.ietltd.com Proudly serving laboratories worldwide since 1979 CALL +1.847.913.0777 for Refurbished & Certified Lab Equipment ABI Q Trap Pro LC/MS/MS Accurate, High-Throughput Protein Identification
More informationInformation Dependent Acquisition (IDA) 1
Information Dependent Acquisition (IDA) Information Dependent Acquisition (IDA) enables on the fly acquisition of MS/MS spectra during a chromatographic run. Analyst Software IDA is optimized to generate
More informationHR/AM Targeted Peptide Quantification on a Q Exactive MS: A Unique Combination of High Selectivity, High Sensitivity, and High Throughput
HR/AM Targeted Peptide Quantification on a Q Exactive MS: A Unique Combination of High Selectivity, High Sensitivity, and High Throughput Yi Zhang 1, Zhiqi Hao 1, Markus Kellmann 2 and Andreas FR. Huhmer
More informationSILAC and TMT. IDeA National Resource for Proteomics Workshop for Graduate Students and Post-docs Renny Lan 5/18/2017
SILAC and TMT IDeA National Resource for Proteomics Workshop for Graduate Students and Post-docs Renny Lan 5/18/2017 UHPLC peak chosen at 26.47 min LC Mass at 571.36 chosen for MS/MS MS/MS MS This is a
More informationPosterREPRINT COMPARISON OF PEAK PARKING VERSUS AUTOMATED FRACTION ANALYSIS OF A COMPLEX PROTEIN MIXTURE. Introduction
Introduction The study of protein expression allows a greater understanding of biological function and can now routinely be performed using mass spectrometry. However, analysis of the post-translational
More informationMass spectrometry has been used a lot in biology since the late 1950 s. However it really came into play in the late 1980 s once methods were
Mass spectrometry has been used a lot in biology since the late 1950 s. However it really came into play in the late 1980 s once methods were developed to allow the analysis of large intact (bigger than
More informationPeptide Targeted Quantification By High Resolution Mass Spectrometry A Paradigm Shift? Zhiqi Hao Thermo Fisher Scientific San Jose, CA
Peptide Targeted Quantification By High Resolution Mass Spectrometry A Paradigm Shift? Zhiqi Hao Thermo Fisher Scientific San Jose, CA Proteomics is Turning Quantitative Hmmm.. Which ones are my targets?
More informationOptimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics* S
Research Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics* S Wilhelm Haas, Brendan K. Faherty, Scott A. Gerber, Joshua E. Elias, Sean A. Beausoleil, Corey E. Bakalarski,
More informationDIA-Umpire: comprehensive computational framework for data independent acquisition proteomics
DIA-Umpire: comprehensive computational framework for data independent acquisition proteomics Chih-Chiang Tsou 1,2, Dmitry Avtonomov 2, Brett Larsen 3, Monika Tucholska 3, Hyungwon Choi 4 Anne-Claude Gingras
More informationIdentification and Characterization of an Isolated Impurity Fraction: Analysis of an Unknown Degradant Found in Quetiapine Fumarate
Identification and Characterization of an Isolated Impurity Fraction: Analysis of an Unknown Degradant Found in Quetiapine Fumarate Michael D. Jones, Xiang Jin Song, Robert S. Plumb, Peter J. Lee, and
More informationQuantitation of High Resolution MS Data Using UNIFI: Acquiring and Processing Full Scan or Tof-MRM (Targeted HRMS) Datasets for Quantitative Assays
: Acquiring and Processing Full Scan or Tof-MRM (Targeted HRMS) Datasets for Quantitative Assays Mark Wrona, Jayne Kirk, and Yun Alelyunas Waters Corporation, Milford, MA, USA APPLICATION BENEFITS Ability
More informationIdentification of proteins by enzyme digestion, mass
Method for Screening Peptide Fragment Ion Mass Spectra Prior to Database Searching Roger E. Moore, Mary K. Young, and Terry D. Lee Beckman Research Institute of the City of Hope, Duarte, California, USA
More informationEffective desalting and concentration of in-gel digest samples with Vivapure C18 Micro spin columns prior to MALDI-TOF analysis.
Introduction The identification of proteins plays an important role in today s pharmaceutical and proteomics research. Commonly used methods for separating proteins from complex samples are 1D or 2D gels.
More informationKey Words Q Exactive, Accela, MetQuest, Mass Frontier, Drug Discovery
Metabolite Stability Screening and Hotspot Metabolite Identification by Combining High-Resolution, Accurate-Mass Nonselective and Selective Fragmentation Tim Stratton, Caroline Ding, Yingying Huang, Dan
More informationSERVA ICPL Kit (Cat.-No )
INSTRUCTION MANUAL SERVA ICPL Kit (Cat.-No. 39230.01) SERVA Electrophoresis GmbH Carl-Benz-Str. 7 D-69115 Heidelberg Phone +49-6221-138400, Fax +49-6221-1384010 e-mail: info@serva.de http://www.serva.de
More informationLogViewer: A Software Tool to Visualize Quality Control Parameters to Optimize Proteomics Experiments using Orbitrap and LTQ-FT Mass Spectrometers
COMMUNICATION LogViewer: A Software Tool to Visualize Quality Control Parameters to Optimize Proteomics Experiments using Orbitrap and LTQ-FT Mass Spectrometers Michael J. Sweredoski, Geoffrey T. Smith,
More informationAnalytical determination of testosterone in human serum using an Agilent Ultivo Triple Quadrupole LC/MS
Application Note Clinical Research Analytical determination of testosterone in human serum using an Agilent Ultivo Triple Quadrupole LC/MS Authors Yanan Yang 1, Victor Mandragon 2, and Peter Stone 1 1
More informationWorkflow concept. Data goes through the workflow. A Node contains an operation An edge represents data flow The results are brought together in tables
PROTEOME DISCOVERER Workflow concept Data goes through the workflow Spectra Peptides Quantitation A Node contains an operation An edge represents data flow The results are brought together in tables Protein
More informationRelative quantification using TMT11plex on a modified Q Exactive HF mass spectrometer
POSTER NOTE 6558 Relative quantification using TMT11plex on a modified mass spectrometer Authors Tabiwang N. Arrey, 1 Rosa Viner, 2 Ryan D. Bomgarden, 3 Eugen Damoc, 1 Markus Kellmann, 1 Thomas Moehring,
More informationRelative Quantitation of TMT-Labeled Proteomes Focus on Sensitivity and Precision
Relative Quantitation of TMT-Labeled Proteomes Focus on Sensitivity and Precision R. Viner 1, M. Scigelova 2, M. Zeller 2, M. Oppermann 2, T. Moehring 2 and V. Zabrouskov 1 1 Thermo Fisher Scientific,
More informationAll Ions MS/MS: Targeted Screening and Quantitation Using Agilent TOF and Q-TOF LC/MS Systems
All Ions MS/MS: Targeted Screening and Quantitation Using Agilent TOF and Q-TOF LC/MS Systems Technical Overview Introduction All Ions MS/MS is a technique that is available for Agilent high resolution
More informationTUTORIAL EXERCISES WITH ANSWERS
TUTORIAL EXERCISES WITH ANSWERS Tutorial 1 Settings 1. What is the exact monoisotopic mass difference for peptides carrying a 13 C (and NO additional 15 N) labelled C-terminal lysine residue? a. 6.020129
More informationQuan%ta%on with XPRESS. and. ASAPRa%o
Quan%ta%on with XPRESS and ASAPRa%o 1 Pep%de and Protein Quan%ta%on Raw Mass Spec Data Pep%de Iden%fica%on Pep%de Valida%on Quan%ta%on Protein Assignment Protein List msconvert X!Tandem SpectraST SEQUEST*
More informationAnalysis of Labeled and Non-Labeled Proteomic Data Using Progenesis QI for Proteomics
Analysis of Labeled and Non-Labeled Proteomic Data Using Progenesis QI for Proteomics Lee Gethings, Gushinder Atwal, Martin Palmer, Chris Hughes, Hans Vissers, and James Langridge Waters Corporation, Wilmslow,
More informationOverview - MS Proteomics in One Slide. MS masses of peptides. MS/MS fragments of a peptide. Results! Match to sequence database
Overview - MS Proteomics in One Slide Obtain protein Digest into peptides Acquire spectra in mass spectrometer MS masses of peptides MS/MS fragments of a peptide Results! Match to sequence database 2 But
More informationBioanalytical Chem: 4590: LC-MSMS of analgesics LC-MS Experiment Liquid Chromatography Mass Spectrometry (LC/MS)
Liquid Chromatography Mass Spectrometry (LC/MS) Prelab Questions: Questions to be answered before doing the experiment. The answers are due at the beginning of each experiment without exception (the questions
More informationA Quadrupole-Orbitrap Hybrid Mass Spectrometer Offers Highest Benchtop Performance for In-Depth Analysis of Complex Proteomes
A Quadrupole-Orbitrap Hybrid Mass Spectrometer Offers Highest Benchtop Performance for In-Depth Analysis of Complex Proteomes Zhiqi Hao 1, Yi Zhang 1, Shannon Eliuk 1, Justin Blethrow 1, Dave Horn 1, Vlad
More informationSimplified Approaches to Impurity Identification using Accurate Mass UPLC/MS
Simplified Approaches to Impurity Identification using Accurate Mass UPLC/MS Marian Twohig, Michael D. Jones, Dominic Moore, Peter Lee, and Robert Plumb Waters Corporation, Milford, MA, USA APPLICATION
More informationIdentification of Human Hemoglobin Protein Variants Using Electrospray Ionization-Electron Transfer Dissociation Mass Spectrometry
Identification of Human Hemoglobin Protein Variants Using Electrospray Ionization-Electron Transfer Dissociation Mass Spectrometry Jonathan Williams Waters Corporation, Milford, MA, USA A P P L I C AT
More informationSUPPORTING INFORMATION Antimicrobial activity of chlorinated amino acids and peptides
SUPPORTING INFORMATION Antimicrobial activity of chlorinated amino acids and peptides Melanie S. A. Coker*, Wan-Ping Hu, Senti T Senthilmohan and Anthony J. Kettle Free Radical Research Group, University
More informationElectron Transfer Dissociation of N-linked Glycopeptides from a Recombinant mab Using SYNAPT G2-S HDMS
Electron Transfer Dissociation of N-linked Glycopeptides from a Recombinant mab Using SYNAPT G2-S HDMS Jonathan P. Williams, Jeffery M. Brown, Stephane Houel, Ying Qing Yu, and Weibin Chen Waters Corporation,
More informationImproved Validation of Peptide MS/MS Assignments. Using Spectral Intensity Prediction
MCP Papers in Press. Published on October 2, 2006 as Manuscript M600320-MCP200 Improved Validation of Peptide MS/MS Assignments Using Spectral Intensity Prediction Shaojun Sun 1, Karen Meyer-Arendt 2,
More informationMS-MS Analysis Programs
MS-MS Analysis Programs Basic Process Genome - Gives AA sequences of proteins Use this to predict spectra Compare data to prediction Determine degree of correctness Make assignment Did we see the protein?
More informationOptimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics
MCP Papers in Press. Published on April 23, 26 as Manuscript M5339-MCP2 Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics Wilhelm Haas, Brendan K. Faherty, Scott A. Gerber,
More informationAccelerating the Metabolite Identification Process Using High Resolution Q-TOF Data and Mass-MetaSite Software
Accelerating the Metabolite Identification Process Using High Resolution Q-TOF Data and Mass-MetaSite Software Application ote Drug discovery and development: Metabolite Identifi cation Authors Yuqin Dai,
More informationMultiple Fragmentation Methods for Small Molecule Characterization on a Dual Pressure Linear Ion Trap Orbitrap Hybrid Mass Spectrometer
Application ote: 54 Multiple Fragmentation Methods for Small Molecule Characterization on a Dual Pressure Linear Ion Trap rbitrap Hybrid Mass Spectrometer Kate Comstock, Yingying Huang; Thermo Fisher Scientific,
More informationHOWTO, example workflow and data files. (Version )
HOWTO, example workflow and data files. (Version 20 09 2017) 1 Introduction: SugarQb is a collection of software tools (Nodes) which enable the automated identification of intact glycopeptides from HCD
More informationThe Agilent 6495 Triple Quadrupole LC/MS: Peptide Quantitation Performance
The Agilent 495 Triple Quadrupole LC/MS: Peptide Quantitation Performance Technical Overview Introduction Sample complexity and the low concentration of certain biomarkers are the major challenges encountered
More informationSupplementary Material for: Clustering Millions of Tandem Mass Spectra
Supplementary Material for: Clustering Millions of Tandem Mass Spectra Ari M. Frank 1 Nuno Bandeira 1 Zhouxin Shen 2 Stephen Tanner 3 Steven P. Briggs 2 Richard D. Smith 4 Pavel A. Pevzner 1 October 4,
More informationComprehensive support for quantitation
Comprehensive support for quantitation One of the major new features in the current release of Mascot is support for quantitation. This is still work in progress. Our goal is to support all of the popular
More informationChapter 5. Complexation of Tholins by 18-crown-6:
5-1 Chapter 5. Complexation of Tholins by 18-crown-6: Identification of Primary Amines 5.1. Introduction Electrospray ionization (ESI) is an excellent technique for the ionization of complex mixtures,
More informationAn Effective Workflow for Impurity Analysis Incorporating High Quality HRAM LCMS & MSMS with Intelligent Automated Data Mining
An Effective Workflow for Impurity Analysis Incorporating High Quality HRAM LCMS & MSMS with Intelligent Automated Data Mining Dave Weil, Ph.D. and Jim Lau, Ph.D. Typical Method Conditions: 1260 UHPLC
More informationKey questions of proteomics. Bioinformatics 2. Proteomics. Foundation of proteomics. What proteins are there? Protein digestion
s s Key questions of proteomics What proteins are there? Bioinformatics 2 Lecture 2 roteomics How much is there of each of the proteins? - Absolute quantitation - Stoichiometry What (modification/splice)
More informationYun W. Alelyunas, Mark D. Wrona, Russell J. Mortishire-Smith, Nick Tomczyk, and Paul D. Rainville Waters Corporation, Milford, MA, USA INTRODUCTION
Quantitation by High Resolution Mass Spectrometry: Using Target Enhancement and Tof-MRM to Achieve Femtogram-level On-column Sensitivity for Quantitation of Drugs in Human Plasma Yun W. Alelyunas, Mark
More informationPROTEIN SEQUENCING AND IDENTIFICATION USING TANDEM MASS SPECTROMETRY
PROTEIN SEQUENCING AND IDENTIFICATION USING TANDEM MASS SPECTROMETRY Michael Kinter Department of Cell Biology Lerner Research Institute Cleveland Clinic Foundation Nicholas E. Sherman Department of Microbiology
More informationThermo Scientific LTQ Orbitrap Velos Hybrid FT Mass Spectrometer
IET International Equipment Trading Ltd. www.ietltd.com Proudly serving laboratories worldwide since 1979 CALL +847.913.0777 for Refurbished & Certified Lab Equipment Thermo Scientific LTQ Orbitrap Velos
More informationOverview. Introduction. André Schreiber AB SCIEX Concord, Ontario (Canada)
Quantitation and Identification of Pharmaceuticals and Personal Care Products (PPCP) in Environmental Samples using Advanced TripleTOF MS/MS Technology André Schreiber AB SCIEX Concord, Ontario (Canada)
More informationAnalyst Software. Peptide and Protein Quantitation Tutorial
This document is provided to customers who have purchased AB Sciex equipment to use in the operation of such AB Sciex equipment. This document is copyright protected and any reproduction of this document
More informationTechnical Note. Introduction
Technical Note Analysis and Characterization of Psilocybin and Psilocin Using Liquid Chromatography - Electrospray Ionization Mass Spectrometry (LC-ESI-MS) with Collision-Induced-Dissociation (CID) and
More informationRapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow. LCMS n. Discovery Attribute Sciences
Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMS n Dhanashri Bagal *, Eddie Kast, Ping Cao Discovery Attribute Sciences Amgen, South San Francisco, California, United
More informationProtein Identification Using Tandem Mass Spectrometry. Nathan Edwards Informatics Research Applied Biosystems
Protein Identification Using Tandem Mass Spectrometry Nathan Edwards Informatics Research Applied Biosystems Outline Proteomics context Tandem mass spectrometry Peptide fragmentation Peptide identification
More informationTracking down protein-protein interaction via FRET-system using site-specific thiol-labeling
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2018 Tracking down protein-protein interaction via FRET-system using site-specific
More informationAB SCIEX SelexION Technology Used to Improve Mass Spectral Library Searching Scores by Removal of Isobaric Interferences
AB SCIEX SelexION Technology Used to Improve Mass Spectral Library Searching s by Removal of Isobaric Interferences Differential Mobility Used as a Tool to Address Selectivity Challenges Adrian M. Taylor
More informationIn-gel digestion of immunoprecipitated proteins separated by SDS-PAGE
In-gel digestion of immunoprecipitated proteins separated by SDS-PAGE (Lamond Lab / April 2008)! Perform all the pipetting steps in a laminar flow hood. We routinely do our digestions in our TC room hoods.
More informationMaximizing Triple Quadrupole Mass Spectrometry Productivity with the Agilent StreamSelect LC/MS System
Maximizing Triple Quadrupole Mass Spectrometry Productivity with the Agilent StreamSelect LC/MS System Application Note Authors Kevin McCann, Sameer Nene, Doug McIntyre, Edmond Neo, Dennis Nagtalon, and
More informationApplication Note. Edgar Naegele. Abstract
Fast identification of main drug metabolites by quadrupole time-of-flight LC/MS Measuring accurate MS and MS/MS data with the Agilent 651 Q-TOF LC/MS and identification of main meta-bolites by comparison
More informationNPTEL VIDEO COURSE PROTEOMICS PROF. SANJEEVA SRIVASTAVA
LECTURE-25 Quantitative proteomics: itraq and TMT TRANSCRIPT Welcome to the proteomics course. Today we will talk about quantitative proteomics and discuss about itraq and TMT techniques. The quantitative
More informationSupporting Information
Supporting Information Highly Efficient Ionization of Phosphopeptides at Low ph by Desorption Electrospray Ionization Mass Spectrometry Ning Pan, a, b Pengyuan Liu, b Weidong Cui, c Bo Tang, a Jingmin
More informationAbsolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 42, Issue 1, January 2014 Online English edition of the Chinese language journal Cite this article as: Chin J Anal Chem, 2014, 42(1), 10 15. RESEARCH PAPER
More informationLast updated: Copyright
Last updated: 2012-08-20 Copyright 2004-2012 plabel (v2.4) User s Manual by Bioinformatics Group, Institute of Computing Technology, Chinese Academy of Sciences Tel: 86-10-62601016 Email: zhangkun01@ict.ac.cn,
More informationProudly serving laboratories worldwide since 1979 CALL for Refurbished & Certified Lab Equipment LCQ Deca XP Plus
www.ietltd.com Proudly serving laboratories worldwide since 1979 CALL +847.913.0777 for Refurbished & Certified Lab Equipment LCQ Deca XP Plus Improved Ion Optics for Greater Sensitivity and Precision
More informationMassHunter TOF/QTOF Users Meeting
MassHunter TOF/QTOF Users Meeting 1 Qualitative Analysis Workflows Workflows in Qualitative Analysis allow the user to only see and work with the areas and dialog boxes they need for their specific tasks
More informationTOMAHAQ Method Construction
TOMAHAQ Method Construction Triggered by offset mass accurate-mass high-resolution accurate quantitation (TOMAHAQ) can be performed in the standard method editor of the instrument, without modifications
More informationRapid method development to study plasma stability of diverse pharmaceutical compounds using Rapid Resolution LC and triple quadrupole MS
Rapid method development to study plasma stability of diverse pharmaceutical compounds using Rapid Resolution LC and triple quadrupole MS Application Note Drug Discovery Authors Srividya Kailasam Agilent
More informationA TMT-labeled Spectral Library for Peptide Sequencing
A TMT-labeled Spectral Library for Peptide Sequencing by Jianqiao Shen A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Mathematics
More informationPC235: 2008 Lecture 5: Quantitation. Arnold Falick
PC235: 2008 Lecture 5: Quantitation Arnold Falick falickam@berkeley.edu Summary What you will learn from this lecture: There are many methods to perform quantitation using mass spectrometry (any method
More informationIncreasing Speed of UHPLC-MS Analysis Using Single-stage Orbitrap Mass Spectrometer
Increasing Speed of UHPLC-MS Analysis Using Single-stage Orbitrap Mass Spectrometer Olaf Scheibner and Maciej Bromirski Thermo Fisher Scientific, Bremen, Germany Overview Purpose: Improve the performance
More informationApplication Note LCMS-112 A Fully Automated Two-Step Procedure for Quality Control of Synthetic Peptides
Application Note LCMS-112 A Fully Automated Two-Step Procedure for Quality Control of Synthetic Peptides Abstract Here we describe a two-step QC procedure for synthetic peptides. In the first step, the
More informationQuantitation of TMT-Labeled Peptides Using Higher-Energy Collisional Dissociation on the Velos Pro Ion Trap Mass Spectrometer
Application Note: 520 Quantitation of TMT-Labeled Peptides Using Higher-Energy Collisional Dissociation on the Velos Pro Ion Trap Mass Spectrometer Roger G. Biringer, Julie A. Horner, Rosa Viner, Andreas
More informationThermo Finnigan LTQ. Specifications
IET International Equipment Trading Ltd. www.ietltd.com Proudly serving laboratories worldwide since 1979 CALL +847.913.0777 for Refurbished & Certified Lab Equipment Thermo Finnigan LTQ Specifications
More informationHemoglobin (Hb) exists in the blood cells of
Prediction of Product Ion Isotope Ratios in the Tandem Electrospray Ionization Mass Spectra from the Second Isotope of Tryptic Peptides: Identification of the Variant 131 Gln Glu, Hemoglobin Camden Brian
More informationConc n of A OAs present (um) Conc n of B OAs present (um)
Materials and Instrumentation: Acquity UPLC BEH C18 2.1 x 100 mm, 1.7 µm column, Waters Corporation, Cat. No. 186002352 O-Benzylhydroxylamine, Sigma-Aldrich Cat. No. B22984 N-(3-Dimethylaminopropyl)-N
More informationAnalysis of a Verapamil Microsomal Incubation using Metabolite ID and Mass Frontier TM
Application Note: 320 Analysis of a Verapamil Microsomal Incubation using Metabolite ID and Mass Frontier TM Key Words Metabolism Study Structure Elucidation Metabolite ID Mass Frontier Chromatography
More informationIncreasing the Multiplexing of Protein Quantitation from 6- to 10-Plex with Reporter Ion Isotopologues
Increasing the Multiplexing of Protein Quantitation from 6- to 1-Plex with Reporter Ion Isotopologues Rosa Viner, 1 Ryan Bomgarden, 2 Michael Blank, 1 John Rogers 2 1 Thermo Fisher Scientific, San Jose,
More information