A novel method for multiplex protein biomarker analysis of human serum using quantitative MALDI mass spectrometry

https://doi.org/10.1016/j.jpba.2021.114575Get rights and content

Highlights

  • MALDI MS can simultaneously determine A2M, fetuin-A, and SAA1 concentrations.

  • Clinically correlated concentration limits were identified if possible.

  • Determination of SAA1 concentration permits assessment of systemic inflammation.

  • Fetuin-A is the most significant marker for differentiation of patient groups.

  • Only joint analysis of several factors enables clearer resolution of patient groups.

Abstract

In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4–5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established.

Introduction

Molecular diagnosis of systemic inflammatory response syndrome (SIRS), sepsis, and their possible complications is a very difficult task due to the high variability and non-specific nature of the biomolecules involved in pathogenesis. In the modern literature, about 200 different biomarkers have been described which assist not only differentiation of SIRS and sepsis, but also: guidance of therapy; and forecasting of disease outcome [1]. It is worth noting the role of viruses in the development of systemic inflammation syndrome with severe organ dysfunction, including the development of shock, acute respiratory distress syndrome, disseminated intravascular coagulation, and acute renal failure requiring dialysis. Despite the relatively small share of viral sepsis (in the total number of systemic inflammation cases reported), acute respiratory viral infections, make a significant contribution to the development of complications [2]. These include, in particular: coronaviruses (including the causative agent of COVID-19, SARS-CoV-2); and influenza viruses. Outbreaks caused by these pathogens occur annually, causing massive epidemics with pandemic potential.

Unfortunately, although various biomolecules have been proposed in the literature as markers for SIRS or sepsis, none of their concentrations can currently serve as a unique, reliable indicator for molecular diagnostics. Thus, it seems appropriate for the diagnosis of SIRS and sepsis, as well as for characterization of pathogenesis, to simultaneously measure the concentrations of several biomarkers in one technological solution. Similar attempts have already been made with analysis of the levels of various inflammatory marker combinations, drawn from: alpha-2-macroglobulin (a2m); C-reactive protein (CRP); ferritin; fibrinogen; haptoglobin; procalcitonin; serum amyloids A and P; tissue plasminogen activator; presepsin; and others [3], [4]. At the moment, however, researchers have not been able to get an unambiguous answer to the question of what should be the most concise list of concurrently detected markers of SIRS and sepsis, and what prognostic capabilities it may have.

The main tasks, hoped to be solved by simultaneous measurement of concentrations of several molecular indicators, are: differential diagnosis of sepsis and SIRS of a non-infectious nature; establishment of the nature of the sepsis causative agent (bacterial, fungal or viral infection; mixed infections); estimation of probable outcome; and assessment of the illness severity. All of this information is needed by attending physicians for the timely implementation of adequate therapies and selection of appropriate drugs. The development of new diagnostic test systems that simultaneously measure the levels of several serum biomarkers of inflammation and sepsis is an urgent task for monitoring the condition of hospitalized patients with severe acute respiratory infections (SARI) [5].

In this work, the possibility of simultaneous quantitative assessment, in a single technological solution, of the concentrations of three serum markers of inflammation is considered, namely: alpha-2-macroglobulin (a2m); serum amyloid A1 (SAA1); and fetuin-A (FetA). The feasibility of quantitatively measuring such serum polypeptides using MALDI-MS in principle, using the example of a2m, was earlier shown by the authors [6]. In cases of infectious illness and systemic inflammation, the developed method can be applied to assess the severity of illness course and to guide treatment. It can also provide an independent quantitative assessment of the concentrations of the selected inflammatory markers. This approach enables obtaining quantitative assessment results (biomarker concentrations) faster than standard immunological methods, namely, within approximately 4–5 h. This paper also presents a statistical analysis of cumulative changes in the levels of inflammatory factors, such as FetA and SAA1, as a result of the development of systemic inflammation or in the course of common human viral infections (influenza A, COVID-19).

Section snippets

Human blood serum

The serum bank consisted of 87 samples from: healthy donors (30 patients, as controls); patients with inflammatory soft tissue diseases accompanied by sepsis (17 patients); patients with influenza A (H1N1 or H3N2), confirmed using certified RT-PCR kits for determination of influenza and ARVI (Amplisens®) (20 patients); and patients with PCR-confirmed COVID-19 (20 patients). This serum bank was formed and kindly provided by the Department of Molecular Biology of Viruses of the Smorodintsev

Selection of biomarkers and identification of their fragments

It has been shown previously that addition of trypsin to blood serum leads to a change in its proteolytic activity as a result of the formation of a complex with a2m, namely: trypsin loses its ability to break down high molecular weight proteins, while retaining the ability to break down some small proteins and peptides [6], [9], [27]. It was shown that, as a result of this interaction, a number of stable peptide ions are formed, which have been unambiguously identified as: hemopexin;

Conclusions

This work demonstrates the feasibility of using a quantitative MALDI mass spectrometry method for simultaneous determination of the concentrations of specific inflammatory biomarkers (a2m, fetuin-A, SAA1) in whole blood serum without preliminary fractionation. The method is based on the ability of trypsin, when added to serum, to bind alpha-2-macroglobulin; this leads to a significant change and limitation of trypsin proteolytic activity in favor of lower molecular weight proteins. As a result

Ethics

The study and its protocols were approved by the Smorodintsev Research Institute of Influenza institutional Ethics Committee (protocol No. 108, dated September 03, 2018; protocol No. 164, dated February 12, 2021). Serum samples (control group) were provided by the Blood Transfusion Center, Research Institute of Hematology and Transfusion Science (contract number 128_15092017). Informed consent was obtained from all donors and patients who provided research materials. All biological experiments

CRediT authorship contribution statement

Aleksandr S. Taraskin: Validation, Investigation, Writing – original draft, Konstantin K. Semenov: Formal analysis, Software, Writing – original draft, Alexey A. Lozhkov: Investigation, Irina L. Baranovskaya: Investigation, Aleksandr V. Protasov: Investigation, Edward S. Ramsay: Writing – original draft, Writing – review & editing, Alexandr A. Tyulin: Validation, Investigation, Olga A. Mirgorodskaya: Conceptualization, Andrey V. Vasin: Resources, Sergey A. Klotchenko: Resources, Yana A.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Russian State Assignment for Fundamental Research No 0784-2020-0023 (IL-6, IL-10, INF-λ assays and analyses), by Russian Science Foundation grant No 20-15-00228 (protein biomarker analysis using mass spectrometry), and by a Russian Presidential Scholarship for Young Scientists and Postgraduate Students No SP-5075.2021.5 (data analyses).

References (35)

  • O.A. Mirgorodskaya et al.

    Clinical proteomics: new diagnostic capabilities of mass spectrometry

    Nauchnoe Priborostr.

    (2008)
  • Y.P. Kozmin et al.

    A direct introduction of 18 O isotopes into peptides and proteins for quantitative mass spectroscopy analysis

    Russ. J. Bioorg. Chem.

    (2011)
  • A.V. Protasov et al.

    Quantitative determination of the series of blood serum markers without its preliminary fractionation using the special features of tripsine interaction with alpha-2-macroglobulin using maldi-Ms

    Nauchnoe Priborostr.

    (2019)
  • J.N. Miller

    Using the Grubbs and Cochran tests to identify outliers

    Anal. Methods

    (2015)
  • R. Christensen

    Analysis of Variance, Design, and Regression

    Linear Modeling for Unbalanced Data

    (2016)
  • S. Banik et al.

    Confidence intervals for the population correlation coefficient ρ

    Int. J. Stat. Med. Res.

    (2016)
  • R.E. Chaddock

    Principles and Methods of Statistics

    (1925)
  • View full text