Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.
The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.
Institute of Pharmaceutical Chemistry, Goethe-University, Frankfurt, Germany.
Stavroula
S
Markoutsa
Ute
U
Bahr
Dimitrios G
DG
Papasotiriou
Ann-Kathrin
AK
Häfner
Michael
M
Karas
Bernd L
BL
Sorg
D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*; D013457:Sulfur Compounds / Q000737:chemistry
D006657:Histones; D010455:Peptides; D013388:Succinimides; D013457:Sulfur Compounds; C094773:sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; D001710:Biotin; D001094:Arachidonate 5-Lipoxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine
5-LO phosphorylation; Histone PTMs; MALDI-MS; Sulfo-NHS-SS-biotin derivatization; Technology