मात्रात्मक प्रोटिओमिक्स के साथ एस cerevisiae में प्रोटीन परिसरों की पहचान

Summary

यहाँ हम एक नया Saccharomyces cerevisiae में प्रोटीन परिसरों की पहचान करने के लिए मात्रात्मक प्रोटिओमिक्स तकनीक का वर्णन. इस अध्ययन में, हम SILAC विधि का इस्तेमाल किया है एक साथ आत्मीयता अग्रानुक्रम मास स्पेक्ट्रोमेट्री द्वारा पीछा करने के लिए उच्च विशिष्टता के साथ एक ईआर प्रोटीन, Scs2p के बंधन भागीदारों की पहचान शुद्धि के साथ.

Abstract

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach.

Protocol

सामग्री और तरीके खमीर उपभेदों सभी उपभेदों BY4742 पृष्ठभूमि पर आधारित इस अध्ययन में इस्तेमाल कर रहे हैं. SILAC नियंत्रण तनाव (Mat एक, his3, leu2, ura3, lys2 और arg4: G418): BY4742 (चटाई अल्फा, arg4 विलोपन तनाव (G418: एक his3, ura3, leu2, और arg4 ?…

Discussion

शुद्धि की प्रक्रिया के दौरान बचाया aliquots (1) पूर्व मंजूरी दे दी सेल lysate, (2) बाध्य अंश, (3) अनबाउंड अंश, और (4) eluted अंश शामिल करना चाहिए. हम पश्चिमी द्वारा ऊपर aliquots के प्रोटीन सामग्री का विश्लेषण विरोधी नल एंटीबॉडी या ?…