Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins.
Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.
Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany. jens.spross@pharmazie.uni-halle.de
Jens
J
Sproß
Sebastian
S
Brauch
Friedrich
F
Mandel
Moritz
M
Wagner
Stephan
S
Buckenmaier
Bernhard
B
Westermann
Andrea
A
Sinz
D000818:Animals; D001710:Biotin / Q000737:chemistry*; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D006736:Horses; D036103:Nanotechnology / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003432:Cross-Linking Reagents; D011506:Proteins; D001710:Biotin