The National Cancer Institute, Fort Detrick, Maryland, March 4th and 5th, 2002
Conference Center, Building 549
(Register for Free Workshops at Bottom)
Monday, March 4, 2002 AGENDA
8:00 – 8:30 Sign-In Refreshments Provided
8:30 – 9:00 Dr. Timothy Veenstra Introduction
9:00 – 10:00 Dr. Jim Stevenson Ion Trap Theory
10:00 – 10:15 Morning Break Refreshments Provided
10:15 – 11:00 Dr. Leo Bonilla- Thermo Finnigan’s New Bioworks 3.0
11:00 – 11:45 Dr. Shiaw-Lin Wu- The use of Differential Quantitation in Targeted Proteomics:
Thermo Finnigan Quantitation of ATP receptor (P2X3) in human embryonic kidney cells using Thermo Finnigan New Proteome X with Bioworks 3.0 software and ICAT reagents.
11:45 – 1:00 Lunch – Provided Lunch-Time Seminar (12:15): Dr. Eric Stover, Thermo Hypersil
Interface of Different LC Modes to MS
1:00 – 2:00 Dr. Jeffrey Kowalak 2DLC/MS Application.
2:00 – 3:00 Dr. Donald Hunt Analysis of Differential Gene Expression by Mass Spectrometry
3:00 – 3:15 Afternoon Break Refreshments Provided
3:15 – 4:00 Andreas Huhmer Femtomole Detection Sensitivity of an Automated Sample
Thermo Finnigan Presentation Technique for Peptides and Proteins Using Nanospray Ion-trap Mass Spectrometry.
4:00 – 4:45 Dr. Shiaw-Lin Wu Using MALDI with the Power of an MSn Ion-trap to Quickly
Thermo Finnigan Determine Multiple Peptide’s Sequences and Modification Sites
4:45 – 5:30 Dr. Gary Paul Quantitative and Qualitative Applications of a Novel High
Thermo Finnigan Resolution Triple Quadrupole Mass Spectrometer
5:30 – 6:00 Reception On-Site, Refreshments Provided
Tuesday, March 5, 2002 AGENDA
10:00 – 12:00 Workshop 1 Proteomics using the DecaXP – Dr. Leo Bonilla
12:00 – 1:00 Lunch Provided
1:00 – 3:00 Workshop 2 ICAT Analysis Using Bioworks 3.0 – Dr. Leo Bonilla
In order to provide an accurate amount of refreshments and to register for a workshop on March 5, 2002,
please send an email specifying which workshop (or both) is desired and who will be attending each day to:
The use of Differential Quantitation in Targeted Proteomics: The quantitation of ATP receptor (P2X3) in human embryonic kidney cells by Proteome X with Bioworks software and ICAT reagent.
Shiaw-Lin Wu1, David Barnidge2, Gargi Choudhary1, Leo Bonilla1, Paul Shieh1, and William S. Hancock1
Proteomic Division of ThermoFinnigan1 and Proteomic Division of Neuromics2
The expression level of a given protein in disease state vs the normal state is an important indication in the study of the mechanism and subsequent treatment of the disease. An approach to measuring differential protein expression levels has been published lately by Aebersold et. al. using ICAT reagent with a LCQ ion trap mass spectrometer (Nature Biotechnology Vol 17, pp. 994, 1999). We have demonstrated here a methodology to quantitate the differences of a low level (femtomole) pain-related biomarker protein, ATP receptor (P2X3) in human embryonic kidney cells, by a new LC-MS system (Proteome X) with a new software program (Bioworks) and the ICAT reagent.
Interface of Different LC Modes to MS
Reversed phase LC/ESI/MS is by far the most popular form of LC/MS techniques because a wide range of moderately complex samples can be analyzed at very low levels without molecular weight restrictions. However, this technique also poses a serious challenge because of a need to control the ionization of solutes to optimize the MS interface efficiency (sensitivity) as well as a need to control retention and separation. Although powerful, not all problems can be addressed by this approach. This presentation will include a review of all modes of LC, including reversed phase, normal phase, adsorption, ion exchange and size exclusion with an assessment of how amenable each might be to interface with the MS and how important each might become for various LC/MS applications. Multiple modes or dimensions through the use of valves and stream switching will be mentioned as an on-line strategy for separation of complex mixtures. HPLC column hardware and interface (plumbing) requirements will also be discussed.
Femtomole Detection Sensitivity of an Automated Sample Presentation Technique for Peptides and Proteins Using Nanospray Ion-trap Mass Spectrometry.
Andreas Hühmer1, Helen Tran1, Sally Swedberg1
1Thermo Finnigan, 255 River Oaks Parkway, San Jose, CA 95134.
Nanospray LC ESI-MS/MSn has become an invaluable analytical tool for ultra-sensitive analysis of complex biological samples. Mandated by ever increasing demands for higher sensitivity and limited sample amounts, mass spectrometry detectors are now routinely coupled to micro and nanoscale LC columns for identification and characterization of peptides and proteins. For low-flow LC, where submicroliter flow rates are typical, efficient sample preparation and presentation techniques are crucial to exploit the high sensitivity of mass spectrometry. For example, peptides recovered from in-gel digests need to be desalted and concentrated prior to introduction to LC-MS. For the best experimental results the combination of a peptide trap in line with a capillary column for the automated removal of electrospray incompatible sample components is superior to off-line sample preparation steps. We present method and data that demonstrate the features and advantages of this sample presentation technique for rapid and accurate peptide characterization. We also discuss experimental approaches for data dependent MSn analysis that exploits the unique capabilities of ion-trap MS for the identification and characterization of post-translational modifications in proteins. Different data-dependent acquisition methods are evaluated to maximize sequence coverage and sequence information for those peptide fragments.
Analysis of Differential Gene Expression by Mass Spectrometry
Donald F. Hunt, Departments of Chemistry and Pathology, University of Virginia, Charlottesville, Virginia 22901
Gene expression in bacteria under environmental pressure can now be measured directly by mass spectrometry. Bacillus subtilis has a genome of 4,300 genes and expresses 1,300-1,500 proteins at any given time. To identify genes differentially expressed in the vegetative and sporulation states of the bacterium, samples of cells in both states were lysed separately, proteins were extracted and digested with trypsin, and the …