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.
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http://dx.doi.org/10.1007/s13361-013-0822-7 | DOI Listing |
Anal Chem
December 2024
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven 5600 MB, The Netherlands.
Quantification of intact proteins in serum by liquid chromatography high-resolution mass spectrometry (HRMS) may be a useful alternative to bottom-up LC-MS or conventional ligand binding assays, due to reduced assay complexity and by providing additional information, such as isoform differentiation or detection of post-translational modifications. The 47.2 kDa lung cancer tumor marker neuron-specific enolase γ (NSEγ) was quantified in a clinically relevant concentration range of 6.
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December 2024
Institute of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan.
This research explores the impact of arsenic exposure on serum protein profiles in type 2 diabetes patients, with an emphasis on the AS3MT protein as a biomarker. Utilizing Bradford protein assay, SDS-PAGE, HPLC, and mass spectrometry, we quantified and analyzed variations in serum protein levels, focusing on differences between control groups (82.94 ± 8.
View Article and Find Full Text PDFToxin-producing strains are the etiological agents of the severe upper respiratory disease, diphtheria. A global phylogenetic analysis revealed that biotype gravis is particularly lethal as it produces diphtheria toxin and a range of other virulence factors, particularly when it encounters low levels of iron at sites of infection. To gain insight into how it colonizes its host, we have identified iron-dependent changes in the exoproteome and surfaceome of strain 1737 using a combination of whole-cell fractionation, intact cell surface proteolysis, and quantitative proteomics.
View Article and Find Full Text PDFAnal Chem
December 2024
State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China.
Droplet-based digital PCR has emerged as a powerful platform for nucleic acid-based detection. However, the formation of droplet compartments and the subsequent amplification process in oil present significant drawbacks: instability under harsh thermal conditions, high background fluorescent noise inside droplets, and major difficulty in supporting multistep assays. Alternatively, droplets made of a hydrogel, or other advanced materials, have been adopted and demonstrate promising improvement over conventional droplet-based platforms.
View Article and Find Full Text PDFGlycosylation is one of the most prevalent and crucial protein modifications. Quantitative site-specific characterization of glycosylation usually requires sophisticated intact glycopeptide analysis using glycoproteomics. Recent efforts have focused on the interrogation of intact glycopeptide analyses using tandem mass spectrometry.
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