1. Quantitative Proteomic Profiling by Mass Spectrometry Paolo Lecchi, Ph.D. Dept. of Pharmacology George Washington University Emerging Technologies in Protein Analysis, January 10, 2003

2. Run 2Dgel; stain; scan Excise spot; elute; digest 1000 1500 2000 Mass (m/z) Extract peptides; mass analyze Protein identification 2D-PAGE AND MASS PECTROMETRY... …A PARADIGM IN PROTEOMICS

3. 2D-PAGE HAS SEVERAL LIMITATIONS…alternative multi- dimensional separation schemes have been introduced. Sample: E. coli extract First dimension: y-axis IEF (Biorad rotofor) Second dimension: x-axis IEF-fractions separated by HPLC (reversed phase) Lecchi et. al. JBBM 2002 (in press) IEF-HPLC

4. ... IS NOT JUST ABOUT “SEPARATION POWER” GENES 35,000 PROTEINS 200,000 TRYPTIC PEPTIDES 4,000,000 THERE IS A HUGE DYNAMIC RANGE OF PROTEIN EXPRESSION (12 orders of magnitude)

5. “Proteomics is knowing the structure and function of all proteins from all organisms…that is not possible. We need to be more selective”. George Kenyon, University of Michigan. In his opening remark at the meeting: “Defining the Mandate of Proteomics in the Post-Genomic Era”. National Academies, Washington DC, March 2002.

6. A “selective” method should be able to specifically detect and quantify only those proteins whose levels of expression change... “…don’t waste your time in evaluating proteins that do not change”. DIFFERENTIAL PROTEOMIC PROFILING

7. 2D-PAGE IMAGE ANALYSIS B A Excise spot; elute; digest; extract peptides; MS analyze for protein identification

8. ENRICHED MEDIUM ( 13 C, 15 N) CHEMICAL DERIVATIZATION ( 13 C, 2 H) 18 O WATER ENZYMATIC DIGESTION MS IDENTIFICATION and QUANTITATION A EXTRACTION REDUCTION ALKYLATION DIFFERENTIAL LABELING WITH STABLE ISOTOPES SEPARATION B

9. LC-MS ICAT-D 8 ICAT-D 0 Cells A Cells B Protein extraction Reduction Alkylation Affinity Column Tryptic digestion MIX CHEMICAL LABELING: ICAT strategy

10. Existing methods for differential quantitative proteomics use the relative intensities between the double peaks generated by MS analysis of the two forms (labeled and unlabeled) of a single peptide... ….which peptide ? “…they work just fine if you know in advance what you are looking for”.

11. NF 3 Helium 12 CF 4 ( 13 CF 4 ) 32 SF 6 ( 34 SF 6 ) 1 HF ( 2 HF) Helium NF 3 from HPLC to MS CHEMICAL REACTION INTERFACE CnHnOnNnSnPrCnHnOnNnSnPr PF 5

12. CRIMS HPLC Protein extraction + labeled Cells unlabeled Cells Labeled Cells +Stimulus Metabolic labeling for HPLC-CRIMS analysis E. Coli growing in M9 medium with glucose (either labeled or unlabeled) as the only source of carbon. Stimulus = IPTG 400  M

13. not-stimulated (-)Stimulated (+) Enrichment trace = 13 C trace - 12 C trace x (IR)

14. Differential proteomics by HPLC-CRIMS ENRICHMENT TRACES CARBON TRACE

15. FIRST SEPARATION TECHNIQUE CHEMICAL REACTION INTERFACE MS QUANTITATIVE ISOTOPIC MONITORING MS identification splitter SECOND SEPARATION TECHNIQUE Sample

16. CRIMS FOR PROTEOMIC allows:: Comprehensive evaluation: absolutely no need for any a priori assumptions High precision: we estimated an ability to evaluate any change in protein expression with a precision as great as ~1% High sensitivity: a limit of detection in the femtomole range Wide dynamic range of analysis: linearity on quantitation over three orders of magnitude Selectivity: the possibility to detect one part of a differentially expressed protein against 1000 parts of interfering materials (e.g. present in the same chromatographic peak) …...no need of high resolution chromatography.

17. 2-D PAGE SEPARATION MASS SPEC. IDENTIFICATION AVAILABLE SCHEMES FOR DIFFERENTIAL PROTEOMIC PROFILING MULTIDIMENSIONAL SEPARATION IRMS IDENTIFICATION AND QUANTITATION MULTIDIMENSIONAL SEPARATION MS IDENTIFICATION AND QUANTITATION STABLE ISOTOPE LABELING