Identification of p65-associated phosphoproteins by mass spectrometry after on-plate phosphopeptide enrichment using polymer-oxotitanium films.
Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.
Wang
Wei-Han
WH
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Palumbo
Amanda M
AM
Tan
Yu-Jing
YJ
Reid
Gavin E
GE
Tepe
Jetze J
JJ
Bruening
Merlin L
ML
D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D019295:Computational Biology; D006801:Humans; D047468:Immunoprecipitation; D007948:Leukemia, Monocytic, Acute; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014025:Titanium / Q000737:chemistry; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism
D002457:Cell Extracts; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011108:Polymers; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D014025:Titanium; D014357:Trypsin