Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.

Analogs of substance P (H-RPKPQQFFGLM-NHThe substance P (H-RPKPQQFFGLM-NH 2 ) NK-1 receptor is a member of the class I family within the superfamily of Gprotein-coupled receptors. The NK-1 receptor has been cloned in several species, among them the murine, the rat, and the human. The hNK-1 1 receptor is composed of 407 amino acids organized in 7 membrane-spanning segments. As for all other receptors from this superfamily, the molecular basis of the binding of the preferred endogenous ligand SP to the NK-1 receptor is far from being well understood. Structure-activity relationships in SP (amino acid substitutions or modifications) (1) as well as in the NK-1 receptor (site-directed mutagenesis, construction of deleted or chimeric receptors) (2) have been studied extensively. Beside these studies, a complementary and more direct approach to identify the key points in the interaction between a peptide and a protein is photoaffinity labeling (3).Several photoaffinity-labeling studies of the NK-1 receptor have already been reported (see Fig. 3 To define further the binding site for SP within the NK-1 receptor, we have synthesized photoreactive analogs, [BAPA 0 , (p-Bzl)Phe x , Met(O 2 ) 11 )]SP, incorporating a biotinyl sulfone amino pentanoic acid (BAPA) derivative at the N terminus and the photoreactive amino acid (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence of SP. We previously showed that the analog modified at position 7 has no affinity (M) for the NK-1 receptor (6). The peptide analogs of SP photoreactive at position 4, 5, 6, 9, or 10 have high affinity for the two binding sites associated with the NK-1 receptor transfected in chinese hamster ovary cells and are potent agonists in activating both phosphatidylinositol hydrolysis and cAMP accumulation (11).In this study, we show that these photoreactive analogs also give good yields of photolabeling of the NK-1 receptor transfected in chinese hamster ovary cells. We have used a methodology of photolabeling already described to identify the site of photoinsertion of SP analogs photoactivable at position 8, in the NK-1 receptor (6, 7). This methodology is based on the use of streptavidin-coated magnetic beads to purify the covalent ligand/receptor complex after enzymatic and/or chemical digestions and subsequent analysis by MALDI-TOF MS to identify the site of photoinsertion within the receptor (6, 7). EXPERIMENTAL PROCEDURES Materials

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.

Objective-Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor.However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. Methods and Results-A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. Conclusion-This