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

Mieke Metzemaekers,Paul Proost,Barbara Neerinckx,Patrick Verschueren,Anneleen Mortier,Sara Abouelasrar Salama,Erik Martens,Mieke Gouwy,Sofie Vandendriessche,Jennifer Vandooren,Carine Wouters,Rik Janssens,Eva Ganseman,Lien De Somer,Jo Van Damme,Ghislain Opdenakker,Sofie Struyf

doi:10.3389/fimmu.2021.644725

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.

Highlights

Neutrophils are the most abundant leukocyte type in human blood (1)
Mature secreted chemokines are susceptible to site-specific proteolysis, citrullination, nitration and glycosylation, with modificationand ligand-dependent consequences for their biological functions [reviewed in (6, 9–11)]
Elution was performed with an acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid (TFA), with two percent of the effluent being used for analysis with online electrospray ionization – ion trap mass spectrometry (AmaZon SL mass spectrometer; Bruker Daltonics, Bremen, Germany)

Summary

Introduction

Neutrophils are the most abundant leukocyte type in human blood (1). These innate immune cells are endowed with a comprehensive anti-microbial machinery and are usually the first responders to infection or tissue injury (2). Chemokines play a central role in this process (3). These low molecular mass proteins function primarily by activation of dedicated heptahelical G proteincoupled receptors (GPCRs) (4, 5). The precise chemokine activity and availability in vivo are the labyrinthine outcome of multiple regulatory mechanisms with a significant role for Quantification of CXCL8 Proteoforms posttranslational modifications (6–9). Mature secreted chemokines are susceptible to site-specific proteolysis, citrullination, nitration and glycosylation, with modificationand ligand-dependent consequences for their biological functions [reviewed in (6, 9–11)]

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Conclusion