Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.
Chemical cross-linking combined with mass spectrometry (MS) has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine-reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them to enrich cross-linked peptides. The reaction of biotin-tagged cross-linkers with purified HIV-1 CA resulted in the formation of hanging and intramolecular cross-links. The peptides modified with biotinylated cross-linkers were effectively enriched and recovered using a streptavidin-coated plate and MS-friendly buffers. The enrichment of modified peptides and removal of the dominantly unmodified peptides simplify mass spectra and their analyses. The combination of the high mass accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS and the tandem mass spectrometric (MS/MS) capability of the linear ion trap allows us to unambiguously identify the cross-linking sites and additional modification, such as oxidation.
Kang
Sebyung
S
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-2170, USA.
Mou
Liyuan
L
Lanman
Jason
J
Velu
Sadanandan
S
Brouillette
Wayne J
WJ
Prevelige
Peter E
PE
D001710:Biotin / Q000138:chemical synthesis / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry / Q000379:methods*
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D001710:Biotin