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.