From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

Abstract

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography—tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and –activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

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“…Such a method is essential, as the proteoforms cannot be distinguished by standard immunoassays like ELISAs or western blot since (a) available antibodies fail to discriminate between the proteoforms and (b) western blot cannot detect minor differences in molecular mass due to the subtle proteolysis. With this technique, all the natural CXCL8 proteoforms were found in the synovial fluids of arthritic patients [ 98 ]. Since proteases truncating CXCL8 are usually upregulated in inflammatory conditions, detecting different CXCL8 proteoforms with highly differential activity in samples from patients with inflammatory diseases will be relevant.…”

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“…The 69 aa form is most active in in vitro chemotactic assays. 28) There are four cysteine residues forming two disulfide linkage (cysteine 7-cysteine 34 and cysteine 9-cysteine 50) and 14 basic aa residues (lysine and arginine), which confer the basicity required for the binding of IL-8 to heparan sulfateproteoglycan without N-glycosylation sites. 29) G.M.…”

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“…However, this is currently impractical given the batch testing requirement of commercial kits, which have been designed for research purposes only rather than clinical practise. More recent studies have shown the quantification of cytokines and chemokines is moving towards proteomic analysis using tandem mass spectrometry, 63 , 64 , 65 , 66 which provides a promising future opportunity.…”