Quantification of intact covalently metal labeled proteins using ESI-MS/MS.
Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.
Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, Germany.
David
D
Benda
Gunnar
G
Schwarz
Sebastian
S
Beck
Michael W
MW
Linscheid
D000328:Adult; D006573:Heterocyclic Compounds, 1-Ring; D006801:Humans; D007460:Iodoacetamide; D007553:Isotope Labeling / Q000379:methods*; D028581:Lanthanoid Series Elements; D008297:Male; D008670:Metals / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D006573:Heterocyclic Compounds, 1-Ring; D028581:Lanthanoid Series Elements; D008670:Metals; D011506:Proteins; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D007460:Iodoacetamide
lanthanide DOTA; mass spectrometry; metal labeling; quantitative proteomics; stable isotope labeling