CPSA Digest 2002

Proceedings -Tuesday, October 8, 2002

TuOD3

Unraveling Signaling Pathways Using Mass Spectrometry

Background
Current advances in biology have been influenced by sequencing of the human genome. However, knowledge of DNA structure is only the beginning to understanding protein chemistry. The task of protein sequencing has now become an experiment in protein identification. Once sample preparation is completed, a protein can be identified in under an hour if its sequence is previously contained within a database. This improvement in the speed with which proteins can be identified is due in large part to advances in mass spectrometry (MS). Using MS, low femtomole amounts of peptides produced from enzymatic digestion of SDS-PAGE derived bands are sufficient to identify proteins from a database. Using this facility in combination with gene tagging techniques and one- or two-step affinity purification protocols makes it possible to identify the composition of protein complexes and identify enzyme substrates. This presentation discusses how various signaling pathways have become unraveled, or determined, using mass spectrometry techniques.

Premise
It is known that proteins do not function alone within cells. They all exist in stable or mostly as transient protein complexes and these complexes perform the work of running the cell. If a gene of unknown or poorly characterized function can be isolated with one or more of its interaction partners, a biological function can then be suggested. Proteins that co-purify can tentatively be assigned to the same biological pathway or pathways. Alternatively, since many proteins are likely to be highly multi-tasking, isolating the uncharacterized protein under differing conditions may provide information on a different set of interacting proteins. Functional proteomics can then be described as the "identification of protein interaction partners," as illustrated below. Here, items interacting with a tag can be co-purified along with that tag under non-denaturing conditions.

One area of interest within GSK is phagocytosis. In this process, a surface receptor of a cell recognizes a foreign particle. The membrane opens up, swallows that particle and internalizes it. Early steps in phagocytosis involve tyrosine phosphorylation of receptors and adapter proteins via the Src family of tyrosine kinase enzymes. Which proteins bind and to which sites? The identification of SH3 domain ligands for the Src family kinase hck was discussed in detail; briefly, a GST- Hck SH3 fusion protein was used to make an Hck SH3 domain affinity column. Protein lysate from 800 mL of phorbol ester-treated U937 cells was incubated with the GST-Hck SH3 resin. Hck SH3 binding proteins were then eluted and separated by SDS-PAGE. Hck is known to be involved in Fc gamma RII mediated phagocytosis in monocytes. ELMO1 has been shown to be associtated in a Crk11/Dock180/ELMO1 complex. The interaction of Hck and ELMO1 raises the possibility that the ELMO1 complex may play a role in this process and that the regulation of the complex may be phosphorylation dependent.

Protein phosphorylation controls many signaling pathways which regulate cell growth, metabolism, motility and differentiation. Protein phosphorylation is catalyzed by a large, diverse group of protein kinases. Phosphorylation of intracellular proteins plays an essential role in signal transduction.

Methods for studying protein phosphorylation need to highly sensitive and highly selective. Many regulatory phosphoproteins are expressed in low abundance. In addition, most phosphoproteins are phosphorylated on more than one site and utilization at any given site is often sub-stoichiometric.

The most common phosphorylation mapping strategy utilizes protein isolated from 32P-labelled cells followed by tryptic digestion and thin layer chromatography. Follow-up involves extraction of the phosphopeptide spot, phosphoamino acid analysis, deductive reasoning, and "silent" Edman sequencing of 32P-labelled peptides. The method is highly sensitive Non-trivial, time consuming, labor intensive. Especially unappealing to contemplate isolation of low copy substrates from large cultures of 32P-labelled cells.

A capillary LC-MS system for multidimensional phosphopeptide mapping was implemented and used for this analysis, as shown below.

Overall, some conclusions from this work can be stated as follows:

• Complete phosphorylation site mapping can be accomplished at levels of about 100-200 fmol of phosphopeptide


• The multidimensional MS method described can be used to reliably detect relative changes in the phosphorylation profile of a protein in response to changes in cellular conditions.


• Phosphorylation profiling can probably be accomplished at the 10-25 fmol level.

References

F. Zappacosta, M.J. Huddleston, R.L. Karcher, V.I. Gelfand, S.A. Carr and R.S. Annan. "Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins." Anal Chem (Jul 1) 74 (2002) 3221-3231.

W. Shou, R. Verma, R.S. Annan, M.J. Huddleston, S.L. Chen, S.A. Carr and R.J. Deshaies. "Mapping phosphorylation sites in proteins by mass spectrometry." Methods Enzymol 351 (2002) 279-296.

M.P. Scott, F. Zappacosta, E.Y. Kim, R.S. Annan and W.T. Miller. "Identification of novel SH3 domain ligands for the Src family kinase Hck. Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1." J Biol Chem (Aug 2) 277 (2002) 28238-28246.

R.S. Annan, M.J. Huddleston, R. Verma, R.J. Deshaies and S.A. Carr. "A multidimensional electrospray MS-based approach to phosphopeptide mapping." Anal Chem (Feb 1) 73 (2001) 393-404.

S. Kassis, T. Melhuish, R.S. Annan, S.L. Chen, J.C. Lee, G.P. Livi and C.L. Creasy. "Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue." Biochem J (Jun 1) 348 Pt 2 (2000) 263-272.

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