A Free Proteomics Seminar, Including Two In-Depth Workshops at:

The National Cancer Institute, Fort Detrick, Maryland, March 4^th and 5^th, 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

NCI/FCRF

9:00 – 10:00 Dr. Jim Stevenson Ion Trap Theory

Research Triangle Institute

10:00 – 10:15 Morning Break Refreshments Provided

10:15 – 11:00 Dr. Leo Bonilla- Thermo Finnigan’s New Bioworks 3.0

Thermo Finnigan

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.

National Institute of Mental Health

2:00 – 3:00 Dr. Donald Hunt Analysis of Differential Gene Expression by Mass Spectrometry

University of Virginia

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

CONTACT INFORMATION

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:

ggregory@thermofinnigan.com

AVAILABLE ABSTRACTS

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

Eric Stover

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 resulting mixtures of approximately 60,000 tryptic peptides in each sample were then analyzed separately by nanoflow chromatography and electrospray ionization on a home built Fourier transform mass spectrometer. This latter instrument operates at a resolution of 40,000, measures masses to three decimal places, and detects peptides at the low attomole level (1). Subtractive analysis of the two data sets identified candidate peptides unique to the sporulation state. Sequence information on these peptides was obtained by employing nanoflow chromatography, peak parking technology, and electrospray ionization on an ion trap mass spectrometer (2). Parent proteins were identified by searching the resulting collision activated dissociation mass spectra against the Bacilus subtilus database with SEQUEST software. The above approach has also been employed to identify: (a) gene families upregulated in nutritionally starved E. coli, (b) protein cargo in yeast transported from the cytoplasm to the nucleus by different karyopherins, (c) the 28 protein components that constitute the U3 snoRNP complex, (d) class I peptides expressed uniqely on the surface of prostate cancer vs healthy prostate cells and tuberculosis infected vs normal macrophage cells, (e) differential display of phosphoprotein expression, and (f) differential display of membrane proteins expressed on cancer cells.

1) Sub-Femtomole MS and MS/MS Peptide Sequence Analysis Using LC-Nano-ESI Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, S.E. Martin, J. Shabanowitz, D.F. Hunt, and J.A. Marto, Anal. Chem., 2000, 72, 4266-4274.

2) Sequencing the Primordal Soup, J. Shabanowitz, R.E. Settlage, J.A. Marto, R.E. Christian, F.M. White, P.S. Russo, S.E. Martin and D.F. Hunt, Proceedings of the 4^th International Symposium on Mass Spectrometry in the Life and Health Sciences, San Francisco, CA, August 25-29, 1998, pg 163-177.

Using MALDI with Ion-trap MS-to-N Power to Quickly Determine Multiple Peptide’s Sequences and Modification Sites

Shiaw-Lin Wu, Pavel Bondarenko, Andrian Land, Viatcheslav Kovtoun, George Stafford, Sally Swedberg, Ken Miller, Paul Shieh, and Bill Hancock

Protein identification using peptide mass maps and database searching has become an important technique for proteomic studies. MALDI-TOF MS has been used extensively to identify samples extracted from gel spots by this approach. Also proteins purified from multi-dimensional separation techniques have been characterized by off-line coupling with MALDI-TOF MS. However, MALDI-TOF MS has the disadvantage that MS/MS is better performed on tandem mass spectrometers. The power to fragment selected ions further can be key to assigning the position of modifications as well as determining the structure of the modification. As an alternative, this report described an atmosphere-pressure MALDI source coupled to an ion-trap MS (LCQ-Deca-XP), which was used to assign peptide sequences by both MS and MS/MS spectra. The structure of modifications (e.g. disulfide linkage or phosphorylation) was determined by further fragmentation in this device (e.g. MS to 3 in the ion trap). This new MALDI-Ion Trap is commercially available and compatible with the current Xcalibur control software and Bioworks data-handling software, which allows samples to be run and data to be analyzed automatically.

Quantitative and Qualitative Applications of a Novel High Resolution Triple Quadrupole Mass Spectrometer

Dr. Gary Paul

In this presentation the quantitative and qualitative applications of a new triple quadrupole mass spectrometer, the TSQ Quantum, will be discussed. New instrumental features such as an orthogonal API source, two square quadrupole ion guides, a 90 degree bent collision cell and hyperbolic rods with a larger field radius have led to a much-improved quantitative SRM performance at unit resolution for the TSQ Quantum where analyte sensitivities as low as 1 fg on-column and linear dynamic ranges up to six orders of magnitude have been achieved. In addition, the unique ability of the TSQ Quantum to achieve high resolution mass separation of an analyte from isobaric interferences has often resulted in improved signal-to-noise ratios for low-level analytes housed within dirty biological matrices relative to those obtained at unit resolution by typical triple quadrupole mass spectrometers. Thus, the high resolution capability of the TSQ Quantum can potentially lower limits of analyte quantitation, lead to larger dynamic ranges and improve the accuracy and precision of quantitative measurements at lower analyte concentrations. In short, the ability of the TSQ Quantum to perform high resolution mass measurements has added an extra level of specificity to the SRM experiment. The high resolution capability of the TSQ Quantum also adds an extra dimension in terms of the qualitative applications of a triple quadrupole mass spectrometer. The ability of high resolution to separate co-eluting components which differ in mass by only 0.1 Da and then perform high performance MS/MS on the isolated species allows for the structural elucidation of analytes of interest such as metabolites which can be masked by isobaric impurities present in the biological matrix. In addition, flexible resolution on both Q1 and Q3 allows for charge state determination of parent and product ions for biological molecules of interest. Coupled with traditional triple quadrupole mass spectrometer utilities such as metabolite LC/MS profiling in complex mixtures where precursor and neutral loss scanning LC/MS/MS experiments coupled with data dependency result in the collection of only drug-related product ion scans, the TSQ Quantum has proven to be a powerful tool in the field of structural elucidation in addition to being a high performance quantitation instrument.

kevin.cofield@apg.amedd.army.mil