Comprehensive Characterization of Protein Modifications using Mass Spectrometry and Dry BloodSpots
Sofia Guedes1, Luís Perpétuo2,3, Jacinta Veloso2, Tânia Lima2, Ana F Ferreira3, Inês Pires3, Francisca Savaiva3, André Lourenço3, Liliana Moreira-Costa3, Adelino Leite-Moreira3, Antonio Barros3, Fábio Trindade3, Rui Vitorino1,2,3
1LAQV/REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro
2iBiMED, Department of Medical Sciences, University of Aveiro, Aveiro
3Cardiovascular R&D Centre – UnIC@RISE, Department of Surgery and Physiology, Faculty of Medicine of the University of Porto, Porto, Portugal
Rui Vitorino, iBiMED
e-mail: rvitorino@ua.pt
Department of Medical Sciences
Abstract
Purpose
The main objective of this study is to characterize and analyze modified peptides in DBS samples. This includes deciphering their specific PTMs and understanding their potential impact on the population or disease cohort under study.
Experimental design
Using mass spectrometry-based proteomic approaches, we performed a comprehensive analysis of DBS samples. Our focus was on the identification and quantification of modified peptides. We also took advantage of recent advances in DBS mass spectrometry to ensure accurate detection and quantification.
Results
A comprehensive analysis identified 976 modified peptides in DBS samples. Of these, a subset of 211 peptides was consistently present in all samples, highlighting their potential biological importance and relevance. This indicates a diverse spectrum of PTMs in the proteome of DBS samples.
Conclusions and clinical relevance
Integration of mass spectrometry and proteomics has revealed a broad spectrum of modified peptides in DBS samples and highlighted their importance in biological processes and disease progression. Accurate detection of these PTMs may be critical for risk stratification and disease management. This study improves the understanding of molecular mechanisms underlying biological processes and disease development, providing important insights for clinical applications.
Introduction
Post-translational modifications (PTMs) are essential for regulating the functions, interactions, and localization of proteins, peptides, and lipoproteins. These modifications involve marginal chemical additions to native molecules after their biosynthesis, leading to altered isoforms with modified biological activities[1]. In the field of mass spectrometry-based proteomics, there are two primary approaches for PTM identification and characterization: bottom-up and top-down proteomics. Each approach has its own advantages and disadvantages for the analysis of proteins, protein interaction partners, protein quantification, and PTMs [2]. In bottom-up proteomics, proteins are first enzymatically cleaved into peptides and these peptides are then subjected to MS analysis. The resulting data are used to draw conclusions about the identity, quantity, and modification status of the proteins present in the sample. Bottom-up proteomics has the advantage that it is very sensitive and complex protein mixtures can be analyzed. It is often used for large-scale studies to identify and relatively quantify proteins. However, a disadvantage of this approach is that information about intact proteins can be lost, such as the exact location of PTMs and sequence variations arising from mutations and alternative splicing events[3]. Top-down proteomics, on the other hand, analyzes intact proteins directly rather than their digested peptides. In this approach, all information about the status of the intact protein is preserved, including PTMs and sequence variations. Because the integrity of intact proteins is preserved, top-down proteomics can provide valuable insights into the presence and location of labile PTMs such as phosphorylation[2, 3]. However, due to the greater physiochemical diversity of intact proteins compared to peptides, separating intact proteins on a large scale can be challenging, so traditional top-down studies tend to focus on analyzing single or a few proteins. Both bottom-up and top-down proteomics employ MS /MS techniques to isolate and fragment specific proteoforms of interest. Energetic dissociation methods such as collision-induced dissociation (CID) and high-energy collision dissociation (HCD) are very useful for characterizing protein sequences. However, they tend to cleave bonds with the lowest activation energy, resulting in the loss of labile PTMs and making their localization difficult. On the other hand, non-ergodic fragmentation techniques, including electron capture dissociation (ECD) and electron transfer dissociation (ETD), are particularly useful for the localization of labile PTMs, such as phosphorylation, because they primarily cleave along the protein backbone and retain the labile modifications[3, 4]. PTMs have emerged as significant contributors to the development and progression of various diseases, including cancer, cardiovascular diseases, renal diseases, and metabolic disorders. Despite their clinical relevance as diagnostic markers, the reliable detection and quantification of PTMs remain challenging for current antibody-based clinical diagnostics. However, state-of-the-art mass spectrometric and proteomic approaches offer the necessary precision and resolving power to identify and quantify novel and pathological PTMs[5]. Nevertheless, the translation of these mass spectrometry applications into clinical practice still faces significant obstacles. PTMs have far-reaching clinical implications, as they can modulate enzyme activity, cellular protein localization, protein-protein interactions, and protein degradation. Dysregulation of PTM-dependent protein and lipoprotein modulation is frequently implicated in human diseases. The clinical relevance of PTMs is supported by curated PTM databases that showcase a wide range of modifications associated with various diseases. Enzyme-driven PTMs are essential tools for modulating cellular and protein functions and maintaining cellular homeostasis[6]. The UniProt database currently catalogs over 600 PTMs, encompassing proteins, peptides, and lipoproteins. The identification of an increasing number of PTMs has revealed their involvement not only in disease pathogenesis but also in the aging process[7, 8]. Accurate detection and qualification of these emerging modified biomarkers and mediators are of utmost importance for precise risk stratification of patients and the development of preventive and interventional strategies[9]. PTMs hold significant promise as targets for clinical diagnosis and treatment, and advancements in their detection and quantification will further contribute to the understanding and management of various diseases.
In recent years, there has been a growing number of studies utilizing mass spectrometry with dry blood spots (DBS) to quantify proteins. These studies have successfully identified hundreds of proteins using various types of mass spectrometry techniques. Various quantification methods were used in the included studies, such as MRM (multiple reaction monitoring), LC-MS /MS (liquid chromatography-mass spectrometry), and nano LC-MS /MS. MRM is a targeted approach suitable for quantification of specific proteins or peptides, while LC-MS /MS and Nano LC-MS /MS provide more comprehensive proteome coverage and allow identification of a larger number of proteins. The choice of quantification method may depend on the specific research objectives, such as targeted biomarker validation (MRM) or unbiased protein profile discovery (LC-MS /MS and Nano LC-MS /MS). The number of proteins identified varied considerably between studies. The study by Nieman [10] using nano LC-MS /MS identified the highest number of proteins (714), highlighting the power of this high-resolution technique. Other studies using MRM and LC-MS /MS approaches identified a range of proteins, with the lowest number of proteins (7) identified in the study by Vidoca [11]. The difference in the number of proteins identified may be attributed to the choice of quantification method, sample preparation techniques, and the complexity of the proteome studied. The size of the DBS spot and extraction solution varied among the studies. Eshghi [12] and Vidoca [11] used smaller DBS spots (3 mm and 6 mm, respectively) compared with other studies using spots of 1 cm in diameter. Different extraction solutions such as ammonium bicarbonate, sodium deoxycholate, tris(2-carboxyethyl)phosphine, and urea were used in different studies. The choice of sample size and extraction solution can significantly affect protein extraction efficiency, solubilization, and preservation of post-translational modifications (PTMs).
The incubation time and temperature during sample preparation varied in the studies. Longer incubation times (e.g., 18 hours in Percy’s study[13]) and higher temperatures (e.g., 60 ºC) may improve protein digestion and PTM retention. However, shorter incubation times (e.g., 1 hour in Chambers’ studies[14-16]) may be beneficial for high-throughput analyzes. The choice of incubation conditions should be optimized based on the specific research objectives and the stability of the proteins and PTMs of interest. A limitation of some studies is the lack of detailed information on the specific research context and references for further validation or comparison. Providing references and contextual information would increase the credibility and reproducibility of the results and allow researchers to build on previous work. Overall, the comparative analysis demonstrates the diversity of proteomic approaches, sample preparation methods, and identified protein profiles in DBS-based mass spectrometry studies. The choice of quantification method, sample preparation, and incubation conditions should be carefully considered based on the research objectives and the complexity of the proteome under investigation. In addition, providing comprehensive information and references would facilitate the integration of these results into the broader field of DBS-based proteomics. The aim of this work is to perform a comprehensive characterization of PTMs, including modifications and artifacts, in proteome or peptidome profiling using dry blood spots as a supporting matrix.