Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry.
Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID-MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of (15)N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC-MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of (15)N-rCRP as an internal standard in ID-MS. Standardization of CRP assays should help validate the relationship between CRP and human health.
National Institute of Standards and Technology, Analytical Chemistry Division, Gaithersburg, MD 28099, USA. eric.kilpatrick@nist.gov
Eric L
EL
Kilpatrick
Wei-Li
WL
Liao
Johanna E
JE
Camara
Illarion V
IV
Turko
David M
DM
Bunk
D000595:Amino Acid Sequence; D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D002097:C-Reactive Protein