Isolation and mass spectrometry of transcription factor complexes.
Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary approaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification of three novel subunits.
Sebastiaan Winkler
G
G
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3LD, Hertfordshire, UK.
Lacomis
Lynne
L
Philip
John
J
Erdjument-Bromage
Hediye
H
Svejstrup
Jesper Q
JQ
Tempst
Paul
P
D000123:Acetyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D051548:Histone Acetyltransferases; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D008832:Microchemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000123:Acetyltransferases; D051548:Histone Acetyltransferases