Nature Method Vol 2,No 4, 291

1. In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature Jascha-N Rybak, Anna Ettorre, Brigitte Kaissling, Raffaella Giavazzi, Dario Neri & Giuliano Elia Nature Method Vol 2,No 4, 291

2. The endothelium is highly dynamic structure, morphologically and functionally adapted to meet the unique needs of the underlying tissue. Tumors requires a blood supply for expansive growth, an observation that has stimulated a profusion of research on tumor angiogenesis. Blood vessels of tumors have multiple structure and functional abnormalities. Their unusual leakiness, potential for rapid growth and remodeling, and expression of distinctive surface molecules not only are responsible for mediating hematogenous spread of tumor cells and maintaining the unusual microenvironment of tumors but also are key to the efficacy of targeted tumor therapy. Genes expressed in human tumor endothelium. Science 289, 1197 (2000) Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. American Journal of Pathology 163,1801 (2003) Subtractive proteomic mapping of the endothelial surface in lung and solid tumors for tissue-specific therapy. Nature 429, 629 (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nature Biotechnology 22, 985 (2004)

3. Injections of F9 murine teratocarcinoma Or tumor cells were inoculated in the left kidney Blood components were washed away with perwarmed PBS supplemented with 10% dextran 40 as plasma expander. The perfusion solution contained 1mg/ml sulfo-NHS-LC-biotin in PBS and 10% dextran 40 Quenching step : 50 mM Tris solution in PBS

4. Histochemical analysis of organ and tumor sections skeletal muscle tongue kidney liver Saline Sulfo-NHS-LC-biotin F9 tumor Sulfo-NHS-LC-biotin Anti-CD31 Saline

5. Chromatographic elution profiles of tryptic peptides from the healthy and tumor-bearing kidenys of RENCA mice It is showed that consistent differences among normal and tumor-bearing kidneys

6. Subcellular localization of protein identified Cell surface proteins and extracellular matrix proteins were present in all organs examined, in proportions that ranged cumulatively between 20% and 50%.

8. Western blot analysis of organ-specific markers Anti-Ksp cadherin: anti-kidney-specific cadherin 16 Anti-calcium channel L-type DHPR alpha 1 subunit Anti-calcium channel, voltage-gated alpha2/delta1 subunit Anti-protein tyrosine kinase 7 Anti-T lymphoma invasion and metastasis inducing protein 1

9. Immunofluorescence detection of organ-specific makers kidney Anti-Ksp cadherin Skeletal muscle Anti-calcium channel L-type DHPR alpha 1 subunit Kidney-specific cadherin 16 was detected in lateral cell membranes bordering the intercellular spaces between the tubular epithelial cells. Calcium channel L-type DHPR alpha 1 subunit is seen in the transverse tubule system, surrounding the muscle fibers.

10. This method offers several potential advantages: Is compatible with the use of strong anionic detergents (such as SDS), which are indispensable for the solubilization of most membrane proteins and extracellular matrix components 2. The chemical properties of the reactive organic molecule used in the perfusion reaction can be changed to influence which structures can be labeled and recovered in vivo. Indeed, the approach is not limited to the use of biotin; any other reactive molecule for which a high-affinity reagent is available could be considered. 3. The proteomics analysis described in this article is easy to implement, is sensitive and allows the hundreds of different accessible proteins in the tissues of interest.

11. Transketolase, a thiamine diphosphate-dependent enzyme linking the nonoxidative branch of pentose phosphate pathway to the glycolytic pathway, has been shown to control in vivo tumor growth in mice with Ehrlich ascites tumor and to be overexpressed in highly metastatic adenoid cystic carcinoma cell lines as compared with their poorly metastatic counterparts. Recently, acetyl CoA carboxylase 265 has been identified as a partner of the protein encoded by the breast cancer susceptibility gene BRCA1. This report show that acetyl CoA carboxylase 256 is either more abundant, more easily accessible, or both in the tumors tested than in other normal tissues, suggesting that it could be used as antigen for ligand-based tumor targeting applications. A non-negligible fraction of identified proteins were intracellular proteins or serum components. Sulfo-NHS-LC-biotin has been shown in in vivo studies to be capable of passing through cell membrane and to label intracellular proteins, in addition to surface proteins, but disulfide-linked botin derivatives such as sulfo-NHS-SS-botin yield a more specific labeling of membrane proteins. However, use of sulfo-NHS-SS-biotin did not improve recovery of membrane proteins, possibly owing to in vivo instability. Serum components were observed at higher frequency in solid tumor. • blood coagulation products have long been known as components of provisional stroma that sustains tumor cell growth. • transient thrombotic events in some tumor blood vessels or insufficient perfusion of the tumor mass

12. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry W Andy Tao, Bernd Wollscheid, Robert O’Brien, Jimmy K Eng, Xiao-jun Li, Bernd Bodenmiller Julian D Watts, Leroy Hood & Ruedi Aebersold Nature Method Vol 2,No 8, 591

13. Reversible protein phosphorylation has a vital role in regulating many complex biological processes such as cellular growth, division and signaling. Phosphorylated proteins and, in particular, the dynamically phosphorylated forms of signaling proteins, are often of low abundance. In contrast to identification of phosphorylation sites within these phosphoproteins is far more difficult. To identify the sites of phosphorylation, it is essential to have an efficient strategy for the enrichment of actual phosphopeptides.

14. Several approaches have been explored to date for the selective isolation of phosphopeptides; the most notable of these are either affinity- or chemical derivatization -based. Phosphotyrosine antibody: Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnology 22, 1139 (2004) It is typically limited to specific amino acid sequences or phosphotyrosine-containing species. Immobilized metal ion affinity chromatography: Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature Biotechnology 20, 301 (2002) This approach has yield considerable results, its selectivity appears to be dependent on several operational parameters including the metal ion, the ligand and the stationary phase used. The method appears to be highly dependent on the type of resin and pH condition used for binding, and elution and favors peptides with multiple phosphorylation sites. Chemical modification: A systematic approach to the analysis of protein phosphorylation. Nature Biotechnology 19, 375 (2001) Typically involve several derivatization steps, and to have had limited applications for the analysis of phospho-peptides within complex mixture.

15. 1. Amino protection: t-butyl dicarbonate (tBoc) to eliminate the potential for intra- and intermolecular condensation 2. Condensation reaction: Carbodiimide catalyzes Condensation reactions between the peptides and excess amine to form amide and phosphoamide bonds. 3. Phosphate regeneration: Free phosphate groups are regenerated by brief acid hydrolysis. 4. Condensation and reduction: A carbodiimide-catalyzed condensation reaction attaches cystamine to the phosphate group. Reduction of the internal disulfide of cystamine next generates a free sulfhydryl group. 5. Solid phase capture: The recovered phosphopeptide are attached to a solid phase by reacting the free sulfhydryl groups in the peptides with iodoacetyl groups immobilized on the glass beads. Nature Biotechnology 19, 375 (2001 6. Phosphopeptide recovery: Phosphopeptides are recovered by cleavage of phosphoamidate bonds using TFA at a concentration that also removes the tBoc protection groups. N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide; EDC

16. Schematic illustration of three-step procedure to chemically isolate phosphopeptide 1. Mixtures of peptides are converted to the corresponding methyl esters to protects carboxylate groups. 2. Tagging and isolating phosphopeptides: the methylate peptides are combined and subjected to a one pot reaction in the presence of carbodiimide (EDC), imidazole and a dendrimer. Phosphate groups are readily activated using EDC and imidazole and react with excess amines on the dendrimer to form phosphoramidate bonds. 3. Covalently bound phosphopeptides are readily isolated from nonphosphopeptides using size selective methods such as a simple membrane-based filter device (5 kDa). 4. Phosphopeptides are detached from the dendrimer through a brief acid hydrolysis of the phosphoramidate bonds and isolate using the same membrane-based filter device.

17. Isolation and tagging of phosphopeptides, and validation with beta-casine digests. 100 pmole tryptic digestion of -casine FQS*EEQQQTEDELQDK (2159.8 m/z) sample recovery efficiency: 10 pmloe tryptic peptide from casine were labeled with methyl-d0 and -d3 esterification. Methyl-d0-esterified peptides were subjected to an enrichment step The recovered methyl-d0 phosphopeptide was add into the original methyl-d3 peptide mixtures final yield > 35% of the starting material The phosphorylated peptide was detected as the most prominent peak in the mass spectrum, with little contamination from nonphosphopeptides or other contaminants.

18. Quantification of phosphopeptides in standard protein mixtures Two mixtures consisting of equalmolar quantities of ovalbumin, BSA, beta-lactoglobulin, lysozyme, beta-casine and apomyoglobin (10 pmole each) Each protein mixture was digested and either methyl-d0 or methyl-d3 esterified. Seven carboxylic groups FQS*EEQQQTEDELQDK (2159.8 m/z) Methyl-d0 and -d3 esterification 21Da (10.5 Da for doubly charged ) Light/heavy ratio : 0.89 1091 m/z 1081 m/z

19. Sensitivity of this method for quantitative analysis 500 pmole of BSA digest was spiked with 1 pmole of signle phosphopeptide Phosphorylate angiotensin II (DRVY*IHPF) Mixture was divided equally, and each sample was either methyl-d0 or -d3 esterified. Enrichment LTQ (quadrupole linear ion trap mass spectrometer) 10 fmole of phosphopeptides

20. Analyses of tyrosine phosphorylation sites in human T cells Stimulation of T cells via cell-surface receptors triggers the activation of the TCR and then activates intracellular networks of effector molecules. Dynamic serine/threonine/tyrosine phosphorylation and dephosphorylation of signaling intermediates by kinase and phosphatase, respectively, is critical for the regulation of the T cell signaling network. To fully understand T-cell response, it is necessary to pinpoint temporal phosphorylation and dephosphorylation events in the course of the response of T cells to various stimuli. Jurket T cells Pervanadate (inhibitor of tyrosine phosphatase ) 2 min 10 min Immuno-affinity enrichment (phosphotyrosine antibody : 4G10) sample was either methyl-d0 or -d3 esterified. Enrichment uLC-MS/MS

21. 97 tyrosine phosphoproteins 75 tyrosine phosphorylation sites 80 serine/threonine phosphorylation sites Our two-step approach of enriching tyrosine-phosphorylated proteins by immuno-affinity selection followed by chemical enrichment of phosphopeptides led to the MS detection of all known tyrosine phosphorylated residues in the (ITAMs) of the TCR’s CD3 chians upon pervanadate treatment in a single experiment.

22. Quantitative phosphopeptide analysis In this case, the ratio (light:heavy) was 2.1, indicating that after a 10-min treatment with pervanadate, phosphorylation on two tyrosine residues of this peptide was increased twofold compared to the 2-min treatment.

23. Abundance ratios of tyrosine phosphopeptides isolated from T cells treated with pervandate over 10 min or 2 min. 10 min : Light 2 min : Heavy Ratios: light/heavy Tyrosine phosphorylation increased with increasing time of pervandate treatment. This is indicated by frequent observation of abundance ratios larger than 1.0 for phosphopeptides isolated after treatment for 10 min and 2 min, respectively.

24. This paper describe an alternative general chemical strategy for the enrichment and subsequent mass spectrometric analysis of phosphopeptides, which represents a major advance compared to their previously reported methodology. This improvement arises primarily from directly capturing phosphopeptides in a single-step reaction with a primary amine-containng solution polymer (in this case, a Generation-5 polyamidoamine dendrimer) rather than the complicated chemical transformation described previously. A dendrimer is a perfectly branched, unimolecular solution polymer that has functional groups only at its surface. (polylysine abs polyallylamine may also be suitable for this application) Such soluble reagents permit faster reactions than the chemically functionalized controlled pore glass beads used previously, owing to their higher capacity and the homogenous nature of the reaction medium. Using this approach, the authors identified and quantitated the relative changes in over 150 sites of phosphorylation from Jurket T cells treated with the phosphotase inhibitor. The true value of this work is in the author’s effective adaptation of techniques from other nonbiologically oriented field to solve a biochemical problem. Specifically the use of solution polymers, such as dendrimers, as auxiliaries in organic reactions has a long-established history, but this is the first report of their effective use in a proteomics application.