Quantification of intact and truncated stromal cell-derived factor-1α in circulation by immunoaffinity enrichment and tandem mass spectrometry

J Am Soc Mass Spectrom. 2014 Apr;25(4):614-25. doi: 10.1007/s13361-013-0822-7. Epub 2014 Feb 6.


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.

MeSH terms

  • Animals
  • Chemokine CXCL12 / antagonists & inhibitors
  • Chemokine CXCL12 / blood*
  • Chromatography, Affinity / methods*
  • Dipeptidyl Peptidase 4 / metabolism
  • Macaca mulatta
  • Mice, Inbred C57BL
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Tandem Mass Spectrometry / methods*


  • Chemokine CXCL12
  • Protein Isoforms
  • Dipeptidyl Peptidase 4