PLOS ONE

RESEARCH ARTICLE

Saliva profiling with differential scanning

calorimetry: A feasibility study with ex vivo
samples
Lena Pultrone1☯, Raphael Schmid1☯, Tuomas Waltimo1, Olivier Braissant2,
Monika Astasov-Frauenhoffer ID3*
1 Clinic for Oral Health & Medicine, University Center for Dental Medicine Basel UZB, University of Basel,
Basel, Switzerland, 2 Center of Biomechanics and Biocalorimetry, c/o Department of Biomedical Engineering
(DBE), University of Basel, Allschwil, Switzerland, 3 Department Research, University Center for Dental
Medicine Basel UZB, University of Basel, Basel, Switzerland

☯ These authors contributed equally to this work.

* m.astasov-frauenhoffer@unibas.ch
a1111111111
a1111111111
a1111111111
a1111111111 Abstract
a1111111111
Differential scanning calorimetry (DSC) has been used widely to study various biomarkers
from blood, less is known about the protein profiles from saliva. The aim of the study was to
investigate the use DSC in order to detect saliva thermal profiles and determine the most
OPEN ACCESS
appropriate sampling procedure to collect and process saliva. Saliva was collected from 25
healthy young individuals and processed using different protocols based on centrifugation
Citation: Pultrone L, Schmid R, Waltimo T,
Braissant O, Astasov-Frauenhoffer M (2022) Saliva and filtering. The most effective protocol was centrifugation at 5000g for 10 min at 4˚C fol-
profiling with differential scanning calorimetry: A lowed by filtration through Millex 0.45 μm filter. Prepared samples were transferred to 3 mL
feasibility study with ex vivo samples. PLoS ONE calorimetric ampoules and then loaded into TAM48 calibrated to 30˚C until analysis. DSC
17(6): e0269600. https://doi.org/10.1371/journal.
scans were recorded from 30˚C to 90˚C at a scan rate of 1˚C/h with a pre-conditioning the
pone.0269600
samples to starting temperature for 1 h. The results show that the peak distribution of protein
Editor: Jose M. Sanchez-Ruiz, Universidad de
melting points was clearly bimodal, and the majority of peaks appeared between 40–50˚C.
Granada, SPAIN
Another set of peaks is visible between 65˚C– 75˚C. Additionally, the peak amplitude and
Received: June 23, 2021
area under the peak are less affected by the concentration of protein in the sample than by
Accepted: May 24, 2022 the individual differences between people. In conclusion, the study shows that with right
Published: June 10, 2022 preparation of the samples, there is a possibility to have thermograms of salivary proteins
Copyright: © 2022 Pultrone et al. This is an open that show peaks in similar temperature regions between different healthy volunteers.
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

Data Availability Statement: All relevant data are Introduction

within the paper and its Supporting Information Over the years, body fluids have been providing an excellent base for creating diagnostic tools
files.
as they contain various different proteins and other biomolecules. As blood is circulating
Funding: The authors received no specific funding through all organs—including those with disease—and its collection is well-standardized, that
for this work. makes blood components by far the most common choice for diagnostics [1].
Competing interests: The authors have declared Saliva has foremostly, important biological functions such as lubrification and the cleansing
that no competing interests exist. of the oral cavity, the facilitating of the speech, assistance of the taste, mastication and

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PLOS ONE Saliva profiling with differential scanning calorimetry

swallowing, start of digestion [2]; it also contains a mixture of proteins such as mucins, amy-
lases, defensins, cystatins, histatins, proline-rich proteins, statherin, lactoperoxidase, lysozyme,
lactoferrin, and immunoglobulins [3] that are secreted from multiple salivary glands (parotid,
submandibular, sublingual and other minor glands) [4].
The term “salivaomics” was introduced in 2008 to highlight the rapid development of
knowledge about various “omics” constituents of saliva, including: proteome, transcriptome,
micro-RNA, metabolome, and microbiome. Since then, new technologies and a wide range of
salivary biomarkers have been validated to make the use of saliva a clinical reality [5]. More
than 100 salivary biomarkers (DNA, RNA, mRNA, proteins) in oral cancer detection have
already been identified, e.g cytokines [6]. However, previous studies have confirmed that also
many discriminatory salivary biomarkers can be detected upon the development of systemic
cancers such as pancreatic [7], breast [8], and lung cancer [9].
Spielmann and Wong compared the protein compositions from human salivary and plasma
fluids and found that even though these fluids have less intersection of the same proteins, the
molecular mechanism, biological processes, and cellular elements show similarity [10]. How-
ever, in comparison to blood, saliva has important advantages as a diagnostic fluid: it can be
collected without any help of health professionals [2, 4], in a stress-free non-invasive way with-
out difficulties and many opportunities [11]. This can be crucial for people with mental disor-
ders, children or elderly, where obtaining blood samples can be difficult [12]. Furthermore,
storage and transportation have lower costs [5, 11] and sufficient quantities for analysis are
given, as healthy individuals have a daily salivary secretion of up to 1.5L [13].
Due to the abundance of studies focusing on salivary biomarkers, it is not easy to discover
novel disease markers; however, it is useful to apply different methods that allow possible
detection of changes in the proteome even before clinical signs appear [14, 15]. Garbett et al.
[16] were able to reveal changes in the thermal profiles of major plasma proteins with differen-
tial scanning calorimetry (DSC) analysis from healthy individuals and patients with different
diseases. Indeed, DSC has gone a long way since its development around fifty years ago. In
early studies, mainly large proteins in high concentration were analysed and the focus was pri-
marily on the process of protein folding rather than make use of it in modern medicine [17].
However, information is gained about the thermal stability of the biomolecules as DSC is able
to measure and reveal all small changes in the heat capacity of protein while undergoing tem-
perature changes [18–20]. Now being an elaborate method in research, many different human
proteins are being examined, such as monoclonal antibodies or fibrinogen [21–23]. Moreover,
many studies have concentrated the effort to investigate changes in protein thermograms to be
able to diagnose chronic pulmonary disease [24], type 1 diabetis [25], glioblastoma [26, 27],
melanoma with regional lymph node or distal metastases [28], breast cancer [29], colorectal
cancer [30], and cervical cancer [31]. However, until now, no studies have focused on evaluat-
ing thermograms of protein profiles of saliva by DSC.
These comparisons between saliva and serum and the fact, that saliva is readily available,
should be enough to investigate whether saliva can be used to produce new relevant protein
markers using DSC. Thus, the aim of the study was to determine the most appropriate sam-
pling procedure to collect and prepare saliva and investigate the use DSC in order to detect
saliva thermal profiles of healthy volunteers to evaluate the feasibility of the method.

Results
Although different saliva preparation protocols were used, thermograms were obtained only
with protocol IV, where centrifugation for 10 minutes at 5000 g and filtering through a Millex
filter with a pore size of 0.45 μm were applied. The other protocols resulted in the presence of

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PLOS ONE Saliva profiling with differential scanning calorimetry

bacteria in the saliva sample (average CFU/mL for protocols I-III was 2x103, 3.5x104, and
2.4x103, respectively), or an absence of DSC thermograms due to the loss or degradation of the
proteins (V-VI). Both stimulated and unstimulated saliva was collected as it is to be expected
that unstimulated saliva has a slightly higher concentration of proteins and thus, might lead to
better sample analysis. However, no statistically significant differences were seen in the protein
concentrations between these two groups (average concentration stimulated vs average con-
centration unstimulated; 0.91 ± 0.41 mg/mL vs 0.94 ± 0.37 mg/mL, respectively). Thus, only
stimulated saliva profiles are presented in Table 1 as this is the more convenient way from the
perspective of sample collection for the donors. Parameters for thermal profiles of saliva with
protocol IV are shown in Table 1. The peak distribution was clearly bimodal (Fig 1A) and the
majority of peaks appeared between 40˚C-50˚C. Another set of peaks is visible between 65˚C-
75˚C. No correlations were found between the concentration of proteins and peak temperature
values (r = 0.13, p = 0.34). Additionally, the peak amplitude and area (under the peak) are less
affected by the concentration of protein (r = -0.23, p = 0.09 and r = 0.17, p = 0.19, respectively)
in the sample than by the individual differences between people. Indeed, there was a rather
high variability even in a single volunteer due to the daily variations in the saliva composition
and amount (Fig 1B).
Results for standard proteins obtained are shown in Fig 2 and Table 2. These results are in
line with their known melting temperatures found in the literature. For these protein stan-
dards, the concentration of the protein did show a strong correlation to the parameters of the
thermal profiles. For lysozyme and BSA very good correlations were obtained. The maximum
heatflow correlated well with the concentration (r = -0.99 and r = -0.98 for lysozyme and BSA
respectively–see data in Table 2). Similarly, the enthalpy measured also correlated well (r =
-0.99 and r = -0.99 for lysozyme and BSA respectively–see data in Table 2). For mucin, signal
was much lower and there was a bit more spread in the data measured the correlation between
the peak heatflow measured and concentration was r = -0.94. Also, here a correlation between
the peak heatflow measured and concentration of mucin of r = -0.97 was observed. Overall,
the measurement with standard protein confirms the accuracy and the possible use of TAM48
DSC for such application. All correlation were significant (p<0.05).

Discussion
Blood plasma has been used to detect various diseases based on the overall thermograms deter-
mined by DSC [22, 36]. However, saliva provides a protein profile like this found in blood
plasma and, therefore could be a valuable addition to biomarker collection to differentiate
between healthy and disease. Additionally, collecting saliva samples is of course easier than
having samples of blood from persons; however, that only applies for healthy people with nor-
mal salivary flow. People suffering from dry mouth or other similar conditions might not be
able to provide enough volume to be analysed [2]. Another factor that makes analysing saliva
more complicated than blood products is that saliva contains also of high number of bacteria
and ca 30 times less protein [37]. Moreover, sample preparation includes purification step so
that only the fraction containing salivary, and no bacterial proteins is assessed. Thus, different
protocols were used in this study to evaluate their efficacy on the removal of bacteria. While
only centrifugation was not enough to eliminate the bacteria, excessive filtering on the other
hand led to loss of salivary proteins and no distinctive peaks to be detected in DSC thermo-
grams. In the end the fraction collected by centrifugation followed by one filtering step was the
only one that allowed to obtain thermal profiles from experiment to experiment most likely
due to sufficient amount of proteins.

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PLOS ONE Saliva profiling with differential scanning calorimetry

Table 1. Parameters of thermal profiles obtained from stimulated saliva samples.

Temperature range Peak temperature (˚C) Peak heatflow (μW) Enthalpy (mJ) Concentration (mg/mL)
32.1 -8.4 -15.12 0.87 ± 0.27
30˚C–40˚C

34 -10.53 -17.85 0.95 ± 0.01

39.4 -28.4 -32.55 1.25 ± 0.01
39.4 -23.41 -23.76 0.54 ± 0.01
39.5 -14.28 -21.01 0.54 ± 0.08
39.8 -4.8 -24.3 0.35 ± 0.02
40.3 -11.7 -37 0.40 ± 0.06
40.5 -6.5 -8.6 0.45 ± 0.11
40.9 -11.6 -23.75 0.46 ± 0.01
41 -18.37 -20.09 0.47 ± 0.01
41.1 -17.36 -29.09 0.74 ± 0.01
41.2 -16.1 -25.8 1.17 ± 0.16
41.2 -7.33 -15.2 0.77 ± 0.02
41.3 -8.6 -14.6 1.02 ± 0.02
41.4 -10.74 -26.86 0.88 ± 0.01
41.6 -5.34 -15.2 0.77 ± 0.02
42 -10.8 -11.2 0.91 ± 0.05
40˚C–50˚C

43.6 -8.13 -12.5 0.65 ± 0.02

43.6 -8.68 -11.6 0.95 ± 0.09
43.9 -37.4 -22.9 0.33 ± 0.05
44 -25.4 -39.4 1.37 ± 0.07
45.1 -9.14 -7.15 1.48 ± 0.03
45.1 -4.8 -6.5 0.74 ± 0.07
45.8 -28.3 -54.8 0.44 ± 0.04
46.7 -9.6 -17.3 2.10 ± 0.01
46.9 -13.3 -22 0.83 ± 0.03
47.5 -23.9 -53.4 1.32 ± 0.30
48.1 -27.3 -35.9 0.64 ± 0.05
49 -29.8 -39.5 0.83 ± 0.01
49.5 -24.1 -32 0.81 ± 0.03
50.6 -19.1 -22.9 0.84 ± 0.07
51 -26.4 -40.6 0.81 ± 0.03
1.07 ± 0.50
50˚C–60˚C

51.6 -30.6 -31.7

52.1 -38 -50.7 1.37 ± 0.50
53 -52.5 -69.9 0.66 ± 0.01
53.8 -44.1 -41 1.35 ± 0.03
58 -22.7 -24.2 1.22 ± 0.24
58.8 -25.7 -19.8 1.17 ± 0.16
60.9 -46.8 -134.7 0.69 ± 0.04
62.4 -32.6 -46.2 1.14 ± 0.19
63.8 -90.5 -72.5 1.45 ± 0.29
60˚C–70˚C

64.2 -12.9 -16.5 0.50 ± 0.01

66 -38.45 -26.35 0.68 ± 0.01
68.2 -66.7 -68.6 2.10 ± 0.01
68.3 -22.5 -26 0.56 ± 0.01
69.3 -33.9 -39.6 0.65 ± 0.09
69.8 -21.7 -41.1 0.82 ± 0.10
(Continued )

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PLOS ONE Saliva profiling with differential scanning calorimetry

Table 1. (Continued)

Temperature range Peak temperature (˚C) Peak heatflow (μW) Enthalpy (mJ) Concentration (mg/mL)
70.7 -21.7 -20.7 0.45 ± 0.02
71 -136.06 -91.94 0.71 ± 0.04
70˚C–80˚C

73.5 -20.7 -19.6 0.98 ± 0.01

73.5 -66.8 -40.2 1.14 ± 0.19
74.2 -42.7 -19.3 0.78 ± 0.03
74.5 -42.7 -17.4 0.78 ± 0.03
74.6 -57.2 -49.2 1.13 ± 0.04
76 -93.87 -34.31 1.04 ± 0.18
80.2 -24.1 -26.6 0.47 ± 0.02
80˚C–
90˚C

81.2 -12.2 -11.4 0.49 ± 0.02

https://doi.org/10.1371/journal.pone.0269600.t001

In order to optimise the signal and receive more reliable results from DSC, a higher concen-
tration of proteins in the solution would be desirable. Indeed, compared to plasma where the
average protein concentration ranges between 60–80 mg/mL [38] the range reported for sali-
vary proteins only reaches values comprised between 0.67 to 2.37 mg/mL [39] and only up to
2.1 mg/mL in the present study. It is known that saliva contains only about 0.3% proteins
while over 99% of the solution is water [10]. Therefore, two different protocols (V-VI) were
used to increase the protein concentration in the samples of this study. Unfortunately, the pro-
tocols used here to increase the concentration while decreasing the volume, were not able to
keep the temperature stable enough to avoid protein denaturation. Thus, a more intensive
analysis on how to maintain the integrity of the proteins needs to be assessed maybe by using
buffering system or other protocols for increasing the concentration.
The results reveal that most of the peaks we found were between 30˚C– 50˚C, which corre-
sponds to the knowledge that salivary proteome contains a larger proportion (14.5%) of low
molecular weight proteins, mainly <20kDa. In comparison only 7% of plasma proteome is in
that size range. In total, up to 65% of salivary proteins have a molecular weight under 65kDa,
while in serum that proportion is only 36%. Additionally, there is a fraction of proteins (27%)
with is found common between saliva and plasma, and their molecular weight distributions
are similar to the distributions of the salivary proteome with a tendency toward the low-molec-
ular-weight end, except in the �200 kDa range [40].
Also, the correlation between the protein concentration and parameters assessed by DSC
was in the scope of this study. However, no strong correlation was detected in any of the tem-
perature range groups for the salivary samples. That could be caused by the presence of other
molecules that either interact with protein (stabilizing them) or denaturating or reacting at
same temperature; thus, perturbing the signal. The concentration of the control proteins did
show a strong correlation to the parameters of the thermal profiles; thus, the weak correlation
of saliva samples was not due to the handling or detection, but due to the physico-chemical
properties of the sample. During this project many physico-chemical parameters such as han-
dling time, age of the chemicals, as well as mathematical parameters such as baseline correc-
tion were shown as possible factors that could affect the quality of the data. This was
exemplified by the lower reproducible of the measurement of early measurements of standard
proteins leading to reasonable values but with much higher spread (see S1 Table). Thus, this
should encourage researchers using DSC to use fresh chemicals and to reduced handling time
as much as possible.

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PLOS ONE Saliva profiling with differential scanning calorimetry

Fig 1. Distribution of proteins by denaturation temperature. (A) Peak distribution in saliva samples in 5˚C intervals for all healthy
volunteers tested; (B) DSC pattern from the same healthy volunteer taken different days (colour refers to the sample collected at the same
time; dashed and solid lines show the replicates for a sample collected and treated the same way).
https://doi.org/10.1371/journal.pone.0269600.g001

It is important to also note that although the calorimeter used in this study allows to process
several samples at the same time and was very helpful to establish this proof-of-concept study,
other calorimeters such as nano-DSC or Flash DSC could provide better alternatives using

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PLOS ONE Saliva profiling with differential scanning calorimetry

Fig 2. DSC pattern of increasing concentrations of protein measured in the TAM 48: (A) Mucin, (B) bovine serum
albumin, (C) lysozyme. Peak value and enthalpy measured can be found in Table 2.
https://doi.org/10.1371/journal.pone.0269600.g002

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PLOS ONE Saliva profiling with differential scanning calorimetry

Table 2. Data showing the main DSC peak parameters (enthalpy, peak heatflow and peak temperature) for the standard protein tested.
Concentration [mg/ml] Enthalpy [mJ] Peak heatflow [μW] Peak temperature[˚C] n
BSA 16 -231 ± 20 -18.4 ± 0.7 59.7 ± 0.1 5
BSA 8 -118 ± 14 -11.8 ± 1.2 59.7 ± 0.2 5
BSA 4 -54 ± 14 -5.2 ± 1.0 60.1 ± 0.4 5
BSA 2 -20 ± 7 -2.2 ± 0.5 59.9 ± 0.6 3
BSA [32] 58.8–59.8
BSA [33] 59.8–60.9
Lysozyme 16 -932 ± 7 -33.7 ± 1.5 73.5 ± 0.4 5
Lysozyme 8 -460 ± 8 -17.9 ± 1.7 74.0 ± 0.4 5
Lysozyme 4 -209 ± 5 -9.1 ± 1.8 74.2 ± 0.3 5
Lysozyme 2 -104 ± 2 -5.9 ± 1.1 74.7 ± 0.6 5
Lysozyme [34] -1373 ± 28 NA 73.8 ± 0.1
Lysozyme [35] -1072 ± 6 NA 76.7 ± 0.1
Mucin 64 -37 ± 5 -7.2 ± 1.4 58.7 ± 0.7 6
Mucin 32 -19 ± 4 -4.3 ± 1.2 59.2 ± 0.8 6
Mucin 16 -11 ± 2 -2.5 ± 0.5 59.9 ± 0.8 6
Mucin 8 -5 ± 1 -1.4 ± 0.4 59.7 ± 0.8 3
Saline NA 0±1 -0.9 ± 0.9§ NA 6
§
Indicate the short-term noise rather than a specific peak

https://doi.org/10.1371/journal.pone.0269600.t002

smaller volumes of sample. Additionally, due to their rapid temperature change these instru-
ments can process sample rather fast and still maintaining a good throughput. Moreover, the
saliva samples could benefit from fast scanning rate as it reduces chances of unspecific protein
degradation (by proteases that might be present in the sample).
In conclusion, although saliva is easy to collect, the proteins are very sensitive to tempera-
ture changes before the measurement and thus, an optimal buffering system might be able to
help with this problem and needs to be assessed in more detail. However, the study shows the
first time that thermograms of salivary proteins are showing peaks in similar temperature
regions between different healthy volunteers and DSC could be considered as a method for
further detailed examinations on salivary proteome. Additionally, proteomic data might help
to further assign the peaks observed to proteins or peptides that could eventually later on be
used as biomarkers.

Materials & methods

Preparation of samples
Altogether 25 healthy young volunteers participated in this study (24.9y ± 3.9y). All of them
were verbally informed about the study and upon agreeing to participate, their verbal consent
was registered together with their age. All volunteers were instructed to not eat or drink any-
thing at least 2h prior to donation of unstimulated as well as stimulated saliva (by chewing par-
affin tablets) like it is common practise at a dental check-up. All leftover samples were
discarded by the end of the study. All sample collection performed for this study involving
human volunteers was in accordance with the ethical standards of the institutional and
national research committee and with the 1964 Helsinki declaration and its later amendments
or comparable ethical standards.
All samples were stored constantly on ice. The saliva samples were then prepared using dif-
ferent protocols adapted of knowledge gained through thorough literature search to eliminate

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PLOS ONE Saliva profiling with differential scanning calorimetry

all possible bacterial counterpart from the samples: (I) centrifugation at 6000 g for 20 min at
4˚C followed by centrifugation at 16200 g for 30 min at 4˚C; (II) centrifugation at 20000 g for
30 min at 4˚C; (III) centrifugation at 5000 g for 10 min at 4˚C followed by filtration through
Millex 5 μm filter; (IV) centrifugation at 5000 g for 10 min at 4˚C followed by filtration
through Millex 0.45 μm filter; (V) same as (IV) followed by concentration through Amicon1
Ultra 0.3 mL Ultracel1 membrane; (VI) same as (IV) followed by lyophilization (Integrated
SpeedVac System ISS110, Savant, Fischer Scientific AG, Reinach, Switzerland). All protocols
were screened twice and as only protocol IV revealed reliable results (the other five protocols
did not allow any peaks to be detected), it was repeated for three more times (n = 5).
The concentration of proteins in processed saliva was assessed by Bradford protein assay
and absorption measured at OD595 (Synergy HT Multi-Detection Microplate Reader, Bio-
Tek1, Luzern, Switzerland). Due to the viscosity of saliva samples, they were split into three
aliquots to assure concentration measurements were correct (n = 3).
Standard proteins (bovine serum albumin, mucin from bovine submaxillary glands, lyso-
zyme, paraffin; all from Sigma Aldrich, Buchs, Switzerland) were weighed to match various
concentrations in sterile saline solution as shown in Table 2.

Differential scanning calorimetry

Saliva samples were transferred to 4 mL calorimetric ampoules and then loaded into TAM48
calibrated to 30˚C until analysis. DSC scans were recorded from 30˚C to 90˚C at a scan rate of
1˚C/h with a pre-conditioning the samples to starting temperature for 1 h. Duplicate DSC
scans were obtained for each sample to assure no drop-outs due to single sample failure (for
example: non-optimal closing of an ampoule). Different saliva preparation protocols were
repeated twice altogether thereafter, most optimized protocol was repeated for five times. Stan-
dard protein samples were analysed using the same procedure but with a temperature range
between 50 and 85˚C. All samples were analysed for three parameters: peak temperature (˚C),
peak amplitude (μW), and peak integral (mJ). Data analysis was performed using the manufac-
turer software (TAM assistant), Fityk (https://fityk.nieto.pl/), and with R version 3.4.4. Nor-
mality of all the parameters was checked by the Shapiro-Wilk test for small sample size.
Parametric (Pearson correlation) was used for normally distributed data from samples with
reference compounds (Table 2). Data of healthy volunteers were not normally distributed and
thus, non-parametric test (Spearman correlation) was used to estimate the correlation between
concentration of the samples and the different thermogram parameters. All the statistical anal-
ysis were performed using GraphPad Prism version 9.3.1 for MacOS, GraphPad Software, San
Diego, California USA, www.graphpad.com.

Supporting information
S1 Table. High variation found in measured parameters of thermal profiles obtained for
various standard proteins to verify the suitability of the method that was due to many phy-
sico-chemical parameters such as handling time, age of the chemicals, as well as mathemat-
ical parameters such as baseline correction were shown as possible factors that could affect
the quality of the data.
(DOCX)

Author Contributions
Conceptualization: Tuomas Waltimo, Olivier Braissant, Monika Astasov-Frauenhoffer.
Data curation: Olivier Braissant.

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PLOS ONE Saliva profiling with differential scanning calorimetry

Formal analysis: Lena Pultrone, Raphael Schmid, Olivier Braissant.

Investigation: Lena Pultrone, Raphael Schmid.
Methodology: Olivier Braissant, Monika Astasov-Frauenhoffer.
Project administration: Monika Astasov-Frauenhoffer.
Resources: Tuomas Waltimo.
Software: Olivier Braissant.
Supervision: Tuomas Waltimo, Monika Astasov-Frauenhoffer.
Validation: Olivier Braissant, Monika Astasov-Frauenhoffer.
Writing – original draft: Lena Pultrone, Raphael Schmid.
Writing – review & editing: Lena Pultrone, Raphael Schmid, Tuomas Waltimo, Olivier Brais-
sant, Monika Astasov-Frauenhoffer.

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