Dataset Information

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.

SUBMITTER: Metzemaekers M

PROVIDER: S-EPMC7991300 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

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

Metzemaekers Mieke M Abouelasrar Salama Sara S Vandooren Jennifer J Mortier Anneleen A Janssens Rik R Vandendriessche Sofie S Ganseman Eva E Martens Erik E Gouwy Mieke M Neerinckx Barbara B Verschueren Patrick P De Somer Lien L Wouters Carine C Struyf Sofie S Opdenakker Ghislain G Van Damme Jo J Proost Paul P

Frontiers in immunology 20210311

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 wi ...[more]

PMID: 33777041

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Project description:PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

Dataset Information

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

Publications

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

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