Quantification of intact and truncated stromal cell-derived factor-1α in circulation by immunoaffinity enrichment and tandem mass spectrometry.
Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α's two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions.
Pharmacokinetic Pharmacodynamics and Drug Metabolism, Merck and Co., Inc., Rahway, NJ, 07065, USA, weixun_wang@merck.com.
Weixun
W
Wang
Bernard K
BK
Choi
Wenyu
W
Li
Zhege
Z
Lao
Anita Y H
AY
Lee
Sandra C
SC
Souza
Nathan A
NA
Yates
Timothy
T
Kowalski
Alessandro
A
Pocai
Lucinda H
LH
Cohen
D000818:Animals; D054377:Chemokine CXCL12 / Q000037:antagonists & inhibitors / Q000097:blood*; D002846:Chromatography, Affinity / Q000379:methods*; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D008253:Macaca mulatta; D008810:Mice, Inbred C57BL; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D054377:Chemokine CXCL12; D020033:Protein Isoforms; D018819:Dipeptidyl Peptidase 4