Elsevier

Clinica Chimica Acta

Volume 458, 1 July 2016, Pages 84-98
Clinica Chimica Acta

Review
Challenges in biomarker discovery with MALDI-TOF MS

https://doi.org/10.1016/j.cca.2016.04.033Get rights and content

Highlights

  • MALDI-TOF MS technique is a popular tool in searching human diseases biomarkers.

  • Sample preparation methods for capturing low molecular subproteomes

  • MS data interpretation affected by preprocessing and statistical analysis step.

  • High abundant proteins mostly pointed as potential biomarkers in profiling pattern.

Abstract

MALDI-TOF MS technique is commonly used in system biology and clinical studies to search for new potential markers associated with pathological conditions. Despite numerous concerns regarding a sample preparation or processing of complex data, this strategy is still recognized as a popular tool and its awareness has risen in the proteomic community over the last decade. In this review, we present comprehensive application of MALDI mass spectrometry with special focus on profiling research. We also discuss major advantages and disadvantages of universal sample preparation methods such as micro-SPE columns, immunodepletion or magnetic beads, and we show the potential of nanostructured materials in capturing low molecular weight subproteomes. Furthermore, as the general protocol considerably affects spectra quality and interpretation, an alternative solution for improved ion detection, including hydrophobic constituents, data processing and statistical analysis is being considered in up-to-date profiling pattern. In conclusion, many reports involving MALDI-TOF MS indicated highly abundant proteins as valuable indicators, and at the same time showed the inaccuracy of available methods in the detection of low abundant proteome that is the most interesting from the clinical perspective. Therefore, the analytical aspects of sample preparation methods should be standardized to provide a reproducible, low sample handling and credible procedure.

Introduction

Predictive biomarkers for the diagnosis of cancer, chronic diseases or neurodegenerative disorders are an important and challenging topic in modern medicine [1], [2]. Reliable indicators could facilitate understanding of pathological processes and provide information about cellular status. Their monitoring could contribute to the prevention of disease development. Cell transformation from a normal to a neoplastic one triggers changes in the biological system that may be detected via investigation of human proteome, as proteins and peptides may be abnormally secreted, over/under-expressed, modified or degraded by unusual activation of the proteolytic pathways [3]. An ideal biomarker should increase diagnostic accuracy and enable optimal treatment, improve early detection, be sensitive and specific, be stable and readily available in biological fluids, quantitatively correlate with a disease stage, and be easily identified on a large scale. It is assumed that proteins and peptides involved in a disease development at the molecular level that may serve as potential biomarkers of the disease are present at low concentrations and are hard to detect in body fluids. In human blood, the albumins, transferrins, immunoglobulins, and complement factors account for almost 99% of all proteins, whereas the remaining 1% includes low-abundant circulatory proteins and peptides excreted by healthy as well as by apoptotic and necrotic cells [4].

The “omics” techniques established new trends in systems biology and clinical studies, especially in identification of potential markers connected with pathological states [5], [6]. Thus, global non-specific detection methods are proposed to determine the profiles of phenotypic diseases and non-morbid states by analyzing hundreds to thousands individual compounds reflecting the proteomic changes. Such analyses are possible thanks to cutting-edge mass spectrometry techniques combined with various sample pretreatment methods that provide an accurate characterization of an analyzed sample. A standard strategy used in clinical studies is called a “bottom-up” strategy. Protein composition and expression are compared based on gel separation and staining, which is a method yielding relative quantitative data. The resolved species are digested with an enzyme, usually endopeptidase trypsin, into peptide constituents. Then, identification and quantification are performed based on one or more tryptic peptides corresponding to a unique protein sequence [7]. Despite known limitations of gel separation, such as low reproducibility, inter-sample agreement and time-consuming preparation, over half of the papers published in the years 2000–2010 in the field of proteomics were based on two dimensional gel electrophoresis (2-DE) [8]. Modern combination of mass spectrometry with liquid chromatography (LC) or gel-off systems begins a new era of gel-free proteomics termed as “shotgun”. High speed of an analysis quickly established LC-MS as a central method used by the proteomics research community. Introduction of two-dimensional chromatography (LC/LC) with automated MS/MS mode (MuDPIT, Multi-dimensional Protein Identification Technology) significantly enhanced separation and fragmentation of complex peptide samples in just a single experiment [9]. However, in clinical applications, both identification and quantification are crucial to differentiate between normal and pathological conditions. Therefore, LC-MS combined with labeling or labeling-free strategies may be also useful in the evaluation of relative quantities for the identified proteins. Limitations of this approach include mainly a digestion process with several drawbacks such as an increase in a sample complexity, inter-laboratory variability of up to 60% and preferably lower rate for the digestion for low concentration proteins [7]. Moreover, the proteins need to be fully solubilized and denaturated to be efficiently digested. Alternatively, a ‘top-down” approach is very promising in biomedical and clinical evaluation of disease mechanisms at the systems level. It enables an assessment of all proteoforms with PTMs and sequence variations without enzymatic digestion. However, protein separation methods such as liquid chromatography offer relatively low throughput and a lack of automation possibilities exclude them from large scale analysis [10]. Thus, proteomic studies could provide a broader and a more holistic picture of an analyzed disease. In this review we focus on the application of non-specific biomarkers search based on MALDI-TOF MS (matrix-assisted laser desorption/ionization – time of flight mass spectrometry) profiling pattern. Common sample preparation methods in this field will be discussed in details and their strong and weak points will be highlighted. Moreover, preprocessing of MS data and their statistical analysis, as well as the usefulness of MALDI-TOF/TOF MS in protein identification will be reviewed and their potential in global proteomics clinical investigation will be assessed. Lastly, we will focus on other available LC-MS strategies used in the comparative studies of human samples.

Section snippets

MALDI-TOF MS profiling

Identification of proteome patterns associated with a disease development has become a promising approach in untargeted biomarker studies. Protein and peptide levels present in complex biological fluids may help define the differences between healthy and affected individuals. Therefore, mass spectrometry (MS) profiling combined with sophisticated data mining tools are gaining popularity in clinical proteomic research. Moreover, the strategy reduces the gap between proteomic investigation and

Sample preparation techniques in MALDI-TOF MS

Sample preparation is a crucial step in generating good quality mass spectra that are essential prerequisites for a successful proteomic pattern analysis and that guarantee the largest possible number of peptides. The employed methods are expected to be reproducible, sensitive and selective, to ensure salts and other impurities removal and depletion of high concentration biomolecules that may interfere with the analytical performance. The specimens obtained from patients represent various body

MALDI target preparation

In complex biological samples the competition between co-existing components for desorption and/or ionization processes is known as analyte suppression effect [68]. Therefore, obtaining constant signals for all the investigated analytes in a sample is impossible in direct MALDI analysis. The described sample preparation methods are effective in overcoming this phenomenon. However, additional changes to the standard protocols could improve ion recording in mass spectrum. The detection

Preprocessing and statistical analysis

Many different chemometric approaches used for MALDI-TOF MS spectra processing in biomarker discovery have been presented in the literature [78], [79]. The aim of the preprocessing methods is adequate data preparation that allows for the reduction of the background noise and proper classification of the samples to the studied groups based on statistically significant features selected by chemometric algorithms. Several computational tools suitable in proteomic studies based on MALDI profiling

Identification of m/z based on LC-MALDI-MS/MS system

Identification of selected features in MALDI MS profiles may be useful in gaining biological knowledge about a disease progression and in assessing its clinical significance. This process refers to the attribution of a protein name to one or more MS signals. The profiling methods are mostly involving specific subproteomes in the mass range 1–15 kDa [101]. In complex MS spectra derived from biological samples, the m/z peaks are often effectively detected in the linear mode but poorly in the

MALDI-TOF MS in clinical application

The protein based approach in MALDI-TOF MS profiling has been commercialized and approved for routine use in clinical microbiological laboratories as a tool for identification of microorganisms [142], [143], [144]. Currently, more and more reports are being published on the usefulness MALDI-TOF MS in the diagnosis of infectious diseases [145]. The microorganisms are identified by pattern matching without the need to identify individual peaks. The MS spectrum of unknown microbial isolates is

Other LC-MS/MS based strategies used in clinical proteomics

Liquid chromatography coupled with mass spectrometry (LC-MS/MS) has become the method of choice for quantitative proteomics in clinical analysis [154]. The existing strategies can be divided into two categories, i.e. with and without isotopic labels. The label approach is using covalent tagging for the determination of protein abundance and quantification of up-regulated or down-regulated species under specific conditions. In principle, the peptides derived from various biological samples are

Conclusion and perspectives

This review is focused on MALDI-TOF MS profiling as a potential strategy in clinical proteomics, where the holistic view of a disease development is certainly an emerging need for the modern medicine. Even though the strategy was introduced at the beginning of twenty first century, still numerous challenges as well as further validation and optimization procedures are required to set a uniform workflow for the proteomic community. In particular, the analytical aspects of sample preparation

Acknowledgments

The author, Joanna Hajduk, MSc , received a support for the preparation of her PhD thesis from National Science Centre in Poland within the frame of a doctoral scholarship financing pursuant to the decision No. DEC-2015/16/T/NZ7/00033.The project was supported by National Science Centre, Poland (UMO-2014/15/B/NZ7/00964).

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