Affinity purification of soluble lysosomal proteins for mass spectrometric identification.
This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins (essentially soluble lysosomal proteins) from cells in culture is induced by adding a weak base in the culture medium. Secreted proteins are ammonium sulfate precipitated, dialyzed, and loaded onto the immobilized MPR column. After specific elution and collection of the M6P proteins, these are resolved by either bidimensional or monodimensional gel electrophoresis (designated as 2-DE or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised from the 2-DE gel, or on discrete bands covering altogether the whole length of the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and protein identification is obtained, thanks to peptide mass fingerprints [provided by analysis of the digests by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences (provided by analysis of the digests by the coupling between liquid chromatography and tandem mass spectrometry, LC-MS/MS).
Jaquinod
Sylvie Kieffer-
SK
CEA, DSV, DRDC, Laboratoire de Chimie des Protéines, Grenoble, France.
Chapel
Agnès
A
Garin
Jérôme
J
Journet
Agnøs
A
D002846:Chromatography, Affinity / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008360:Mannosephosphates; D013058:Mass Spectrometry / Q000379:methods; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility; D020298:U937 Cells
D008360:Mannosephosphates; D009363:Neoplasm Proteins; D011506:Proteins; C018589:lysosomal proteins; C027693:mannose-6-phosphate