Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry.
This work demonstrates that amino acid analysis based on isotope dilution mass spectrometry (IDMS) can be applied to quantify proteins having different complexities and natures. Five proteins and one decapeptide were selected for the study: C-reactive protein (CRP), beta-2-microglobulin (B2M), cystatine C (CysC), human serum albumin (HSA), Ara h1, and angiotensin I. The quantification was based on the determination of four amino acids, proline (Pro), isoleucine (Ile), valine (Val), and phenylalanine (Phe) within a working range between 5 and 100 pmol/injection of each amino acid, after 60 min digestion with HCl at 150°C. The amino acids were selected taking into account their abundance in the protein sequence and to include the more difficult to break peptide bonds. Quantification of the protein amounts calculated from each amino acid is consistent, indicating that the method is working reliably. This consistency points to a complete hydrolysis of the proteins. The trueness of the method was proven when dry mass determination after dialysis was applied to HSA and CRP and the results were compared to those from amino acid analysis. Traceability to SI was assured by extensive characterisation of the amino acid calibrants by nuclear magnetic resonance, neutron activation analysis, and Karl Fischer titration.
Muñoz
A
A
Joint Research Centre, Institute for Reference Material and Measurements, Geel, Belgium. amalia.MUNOZ-PINEIRO@ec.europa.eu
Kral
R
R
Schimmel
H
H
D000596:Amino Acids / Q000032:analysis* / Q000302:isolation & purification / Q000592:standards; D000803:Angiotensin I / Q000032:analysis; D052179:Antigens, Plant / Q000032:analysis; D002097:C-Reactive Protein / Q000032:analysis; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015891:Cystatins / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D008565:Membrane Proteins; D010940:Plant Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis
D000596:Amino Acids; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; D015891:Cystatins; D006023:Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; D011506:Proteins; D012709:Serum Albumin; D002097:C-Reactive Protein; D000803:Angiotensin I