Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.
Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact complexes from cell lysates suffers from protein and RNA degradation, loss of transient interactors, and poor overall yields. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol described here has been optimized for the yeast S. cerevisiae.
Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, Canada, H2W 1R7.|Département de biochimie et médicine moléculaire, Faculté de médecine, Université de Montréal, Montréal, QC, Canada, H3T 1J4.
Christian
C
Trahan
Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, Canada, H2W 1R7.
Lisbeth-Carolina
LC
Aguilar
Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, Canada, H2W 1R7. marlene.oeffinger@ircm.qc.ca.|Département de biochimie et médicine moléculaire, Faculté de médecine, Université de Montréal, Montréal, QC, Canada, H3T 1J4. marlene.oeffinger@ircm.qc.ca.|Faculty of Medicine, Division of Experimental Medicine, McGill University, Montréal, QC, Canada, H3A 1A3. marlene.oeffinger@ircm.qc.ca.
Marlene
M
Oeffinger
D002846:Chromatography, Affinity / Q000379:methods; D046911:Macromolecular Substances / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins
Cell lysis; Cryo-milling; Mass spectrometry; Proteomics; Single-step affinity purification; Yeast