Influence of Storage Conditions on MALDI-TOF MS Profiling of Gingival
Crevicular Fluid: Implications on the Role of S100A8 and S100A9 for Clinical
and Proteomic based Diagnostic Investigations
Mariaimmacolata Preianò a,†, Giuseppina Maggisano a,†, Nicola Lombardo b, Tiziana Montalcini b, Sergio
Paduano a, Girolamo Pelaia b, Rocco Savino a and Rosa Terraccianoa,*
a
Department of Health Sciences, Laboratory of Mass Spectrometry and Proteomics, University “Magna
Græcia”, Catanzaro, Italy
b
Department of Medical and Surgical Sciences, University “Magna Græcia”, Catanzaro, Italy
* Address correspondence to Prof. Rosa Terracciano. E-mail: terracciano@unicz.it. Tel.:
++39/09613694085. Fax: ++39/09613694090.
Postal address: Department of Health Sciences, Laboratory of Mass Spectrometry and Proteomics,
University of Catanzaro, University Campus, Europa Avenue, 88100, Germaneto, Catanzaro, Italy.
†
MP and GM equally contributed to this manuscript;
List of abbreviations: GCF, gingival crevicular fluid, SA, sinapinic acid;
Keywords: Biomarkers / Biomedicine / Gingival crevicular fluid / MALDI-TOF-MS / Peptidomics
Subject code
100
90
80
70
60
50
40
30
Subject 1 20
10
w PIC
0
w/o PIC
10000
Subject code
% Intensity
% Intensity
100
90
80
70
60
50
40
30
20
10
0
3300
3360
3420
3480
3540
3600
Subject 1
11000
Mass (m/z)
100
90
80
70
60
50
40
30
Subject 2 20
w PIC
10
w/o PIC
0
10000
15000
Subject code
3360
3420
3480
3540
3600
Subject 2
w PIC
w/o PIC
11000
12000
13000
14000
15000
Mass (m/z)
100
90
80
70
60
50
40
30
Subject 3 20
w PIC
10
0
w/o PIC
10000
Subject code
% Intensity
% Intensity
Subject code
3360
3420
3480
3540
3600
Subject 3
w PIC
w/o PIC
11000
% Intensity
% Intensity
Subject code
Subject 4
3360
3420
3480
Mass (m/z)
12000
13000
14000
15000
Mass (m/z)
Mass (m/z)
100
90
80
70
60
50
40
30
20
10
0
3300
14000
% Intensity
Subject code
Mass (m/z)
100
90
80
70
60
50
40
30
20
10
0
3300
13000
Mass (m/z)
% Intensity
100
90
80
70
60
50
40
30
20
10
0
3300
12000
w PIC
w/o PIC
3540
3600
w PIC
w/o PIC
100
90
80
70
60
50
40
30
20
10
0
10000
Subject code
Subject 4
w PIC
w/o PIC
11000
12000
13000
14000
15000
Mass (m/z)
Figure S1. MALDI-TOF spectra of GCF extracted at time=0, from filter papers by a three steps
centrifugal elution in 2.5% TFA from four healthy subjects. The spectra were acquired in the MW
range from 2000 to 20000 Da using SA as MALDI matrix (1:4 sample-to-matrix ratio in
unsaturated SA 35% ACN in 0.1% TFA). Ranges are shown from 3300 to 3600 and from 10000 to
15000 m/z for the best detection by the readers.
Supplementary Materials and methods
S1. Reagents and Materials
ACN (HPLC grade), water (HPLC grade), TFA (HPLC grade), DTT, iodoacetamide, CHCA and
trypsin from porcine pancreas were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Ammonium bicarbonate was obtained from Fluka (St. Louis, MO, USA). 1D SDS-PAGE was
carried out using a Novex 16% polyacrylamide Tricine Gel (Invitrogen, Carlsbad, CA) on a Xcell
SureLock Mini-Cell (Invitrogen). For 1D SDS-PAGE the protein marker Precision Plus Protein
Dual Color Standards was purchased from BioRad Laboratories (Hercules, CA, USA) and the Color
Marker Ultra-low Range was purchased from Sigma-Aldrich (St. Louis, MO, USA). Coomassie
Brilliant Blue G-250 was obtained from BioRad Laboratories (Hercules, CA, USA). For MS
analysis, external calibration was performed using the 5800 Mass Standards kit (AB SCIEX,
Framingham, MA, USA).
S1. 1D gel electrophoresis and in-gel digestion
1D SDS-PAGE electrophoresis was performed according to Ngo et al. [1] with minor
modifications. Briefly, the samples were heated for 2 min at 85 °C under reducing conditions,
vortex-mixed, and applied to Novex 16% polyacrylamide Tricine Gel (Invitrogen, Carlsbad, CA) on
a Xcell SureLock Mini-Cell (Invitrogen). An aliquot of protein marker (#161-0374) PrecisionPlus
Protein Dual Color Standards BioRad Laboratories Hercules, CA, USA) and color marker (Ultralow Range Sigma-Aldrich, St. Louis, MO, USA) were used on the gels. After staining with
Coomassie Blue Brilliant G-250, protein bands of interest were excised, placed into Eppendorf
tubes, and stored at 4 °C prior to in-gel digestion. Excised bands were destained, dehydrated, and
then incubated overnight at 37°C with modified trypsin (13 ng/μL in 40 mM ammonium
bicarbonate) after reduction and alkylation. Afterwards, the digest solution was acidified with 5µL
of 5% TFA. Peptides from the gel pieces were sequentially extracted three times in 100 μl of 60%
(v/v) ACN, 0.1% (v/v)TFA. The supernatants were combined and dried in a SpeedVac centrifuge.
Dried peptides were then re-suspended in 5 μl of 50% (v/v) ACN, 0.1% TFA for the MALDI-TOF
analysis.
S2. MALDI TOF and MALDI-TOF/TOF Mass Spectrometry
1 μl of the re-suspended digested peptide mixture was mixed with 4 μl of matrix solution (4mg/ml
of CHCA in 50% ACN and 0.1% TFA) and 0.8 μl of the obtained solution was spotted on the
MALDI target plate. The digested peptide mixtures were analyzed in reflector mode using an AB
SCIEX MALDI-TOF/TOF 5800 System (AB Sciex, Framingham, MA, USA) equipped with a
diode-pumped, ND:YLF laser with λ=345 nm wavelength. For MALDI MS measurements in
CHCA the following settings were applied: bin size was set at 1 ns, final detector voltage was 1.980
kV with multiplier value at 0.66. 2000 laser shots were accumulated for each spectrum. MS data
were calibrated via external calibration using the 5800 Mass Standards kit (AB SCIEX,
Framingham, MA, USA) containing des-Arg1-Bradykinin (MH+ 904.4681), Angiotensin I (MH+
1296.6853), Glu-Fibrinopeptide B (MH+ 1570.6774), ACTH (clip 1-17) (MH+ 2093.0867), ACTH
(clip 18-39) (MH+ 2465.1989), ACTH (clip 7-38) (MH+ 3657.9294). Proteins identification was
based on PMF and MS/MS analysis. PMF was performed by comparing the experimentally
determined peptide masses against Swiss Prot sequence database, using Mascot version 2.5.1
(http://www.matrixscience.com/). Up to one missed trypsin cut was allowed and the data were
searched using cysteine carbamidomethylation as fixed modification and methionine oxidation as
variable modification. Candidates with Mascot scores greater than the 95% confidence threshold
(protein score of 65) were accepted. Protein Prospector (http://prospector.ucsf.edu/) was also used
to acquire theoretical masses expected for the digested protein. With regard to the MS/MS
measurements, the voltage settings were 8.0 kV and 15.0 kV for the ion source 1 and source 2,
respectively. Air was used as the collision gas and MS/MS spectra were acquired at a laser energy
setting of 4000-5000. MS/MS data were calibrated against the MS/MS fragments of the m/z
1570.677 Glu-Fibrinopeptide B in the standards. The MASCOT v.2.5.1 search engine
(www.matrixscience.com) was used to compare the TOF/TOF spectra against Homo Sapiens
primary sequence database Swiss-Prot to determine peptide sequence identities. Search parameters
included cysteine carbamidomethylation and methionine oxidation as fixed and variable
modifications respectively; MS tolerance for precursor ions was set at 50 ppm and 0.25 Da for
MS/MS ions. The results of peptide identification are summarized in Table S1.
Table S1: GCF Proteins identified with MALDI-TOF and MALDI-TOF/TOF MS from Gel
bandsa
Protein identified
(Accession Number)
MW (Da)
Sequence coverage
(%)
S100-A8 CalgranulinA (P05109)
10835c
64%
S100-A9
Calgranulin-B
(P06702)
13153 d
81%
Lysozyme C (P61626)
14691e
56%
Experimental monoisotopic [MH]+
of matched peptides (sequence
position) b
863.4 (1-7), 1272.59 (8-18),
963.41 (24-31), 1434.61 (24-35)1,
1562.70 (24-36)2, 1421.61 (37-47),
1549.71 (37-48)1, 950.42 (49-56)1,
822.34 (50-56), 1110.42 (85-93)2,
853.35 (86-92), 982.34 (86-93)1
1806.8451 (11-25), 1455.6276
(26-38), 877.4181 (44-50),
1005.4933 (44-51)1, 1742.7244
(58-72), 1758.7247 (58-72)Ox,
1614.7123 (73-85), 1630.7105
(73-85)Ox, 971.4263 (86-93),
2175.8494 (94-114), 2191.8511
(94-114)Ox
1179.53 (20-28)1, 1325.67 (2939)2, 811.32 (33-39), 827.32 (3339)Ox, 1012.38 (52-59), 981.38
(60-68), 1400.60 (69-80), 942.32
(81-87), 2927.30 (88-115)
a) Proteins identification was based on PMF and MS/MS analysis. Searches were performed under the
following parameters for PMF: Taxonomy, H. Sapiens; Mass Tolerance, 50 ppm; Missing cleavages,
≤ 2; Enzyme, Trypsin; Fixed modifications, Carbamidomethylation; Variable modifications,
Oxidation (M), Charge State, 1+. Search parameters for MS/MS: MS Tolerance, 50 ppm; MS/MS
tolerance, 0.25Da; Enzyme, Trypsin; Charge state, 1+.
b) 1 and 2 superscripts on the m/z values refer to one and two missed cleavages respectively; Ox
superscript refers to oxidation of methionine. Bold type indicates peptide identity verified by
MS/MS.
c) The MW from Swiss Prot is 10885 Da for carbamidomethylation (C) (+57 Da).
d) The MW refers to the protein with these modifications: acetyl (N-term), M missing (N-term) as also
found in Castagnola et al [2].
e) The MW from Swiss Prot is 16982 Da for the presence of peptide signal and for
carbamidomethylation of 8 Cys (C) (+456 Da).
References
[1] Ngo, L. H., Veith, P. D., Chen, Y. Y., Chen, D. et al., Mass spectrometric analyses of peptides
and proteins in human gingival crevicular fluid. J. Proteome Res. 2010, 9, 1683-1693.
[2] Castagnola, M., Inzitari, R., Fanali, C., Iavarone, F. et al., The surprising composition of the
salivary proteome of preterm human newborn. Mol Cell Proteomics 2011, 10, 1-14.
1422
4700 Reflector Spec #1 MC=>NR(2.00)[BP = 1421.6, 19973]
982
% Intensity
822
40
*
20
950
1273
*
*
1110
863
0
800
1435
% Intensity
80
60
960
1120
A
*
1280
Mass (m/z)
1563
*
100
*
*
1550
963
1440
1600
Mass (m/z)
1456
4700 Reflector Spec #1[BP = 1455.7, 15010]
*
100
B
1743
971
% Intensity
60
40
1005
1615 1631
*
1759
% Intensity
80
877
20
0
800
1100
1400
2176
1807
1700
Mass (m/z)
2192
*
2000
2300
Mass (m/z)
4700 Reflector Spec #1 MC=>NR(5.00)[BP = 1455.6, 9396]
1012
100
*
80
942
% Intensity
60 811
827
40
20
0
800
981
850
900
950
Mass (m /z)
C
1000
1050
4700 Reflector Spec #1 MC=>NR(5.00)[BP = 1455.6, 9396]
80
1400
1180
*
60
% Intensity
% Intensity
100
40
20
0
800
1325
1240
2927
1680
Mass (m /z)
2120
2560
*3000
Mass (m/z)
Figure S2. MALDI-TOF mass spectra obtained from in-gel tryptic digestion of S100A8 (Panel A),
S100A9 (Panel B) and Lysozyme C (Panel C). The peptides labeled with asterisks were identified
by MALDI-TOF/TOF MS.
y(2)
4700 MS/MS Precursor 1272.59 Spec #1[BP = 284.1, 1389]
y(3)
543.4
Y(9)
b(10)
Y(8)
y(6)
b(8)
b(7)
y(5)
y(4)
276.2
Y(7)
0
9.0
b(5)
b(2)
b(3)
20
b(1)
40
b(4)
60
ALNSIIDVYHK
b(6)
80
% Intensity
% Intensity
100
810.6
Mass (m/z)
1077.8
1345.0
Mass (m/z)
4700 MS/MS Precursor 1562.7 Spec #1[BP = 110.1, 2823]
337.6
666.2
y(11)
b(9)
b(8)
b(10)
y(6)
b(6)
b(4)
20
0
9.0
b(2)
40
b(5)
60
b(12)
GNFHAVYRDDLKK
80
% Intensity
% Intensity
100
994.8
Mass (m/z)
1323.4
1652.0
Mass (m/z)
4700 MS/MS Precursor 1549.7 Spec #1=>NR(2.00)[BP = 1549.7, 1047]
0
9.0
334.8
660.6
y(8)
y(7)
b(7)
y(3)
20
b(3)
y(2)
40
y(5)
y(6)
y(1)
60
y(11)
KLLETECPQYIR
80
% Intensity
% Intensity
100
986.4
Mass (m/z)
1312.4
1638.0
Mass (m/z)
4700 MS/MS Precursor 822.34 Spec #1=>NR(2.00)[BP = 822.4, 1039]
0
9.0
181.2
353.4
525.6
Mass (m/z)
y(5)
y(4)
b(5)
y(3)
b(4)
y(2)
b(3)
20
y(1)
40
b(2)
60
b(6)
GADVWFK
80
% Intensity
% Intensity
100
697.8
870.0
Mass (m/z)
4700 MS/MS Precursor 982.34 Spec #1=>NR(2.00)[BP = 982.4, 1226]
SHEESHKE
0
9.0
214.8
420.6
Mass (m/z)
626.4
895.41
y(7)
y(6)
b(6)
y(5)
b(5)
y(4)
y(3)
b(4)
20
b(3)
b(2)
40
y(2)
60
% Intensity
% Intensity
80
b(7)
100
832.2
1038.0
Mass (m/z)
Figure S3. MALDI-TOF/TOF spectra with fragment ion signals of peptides obtained from triptic
digestion of S100A8: ALNSIIDVYHK (residues 8 to 18), GNFHAVYRDDLKK (residues 24 to
36), KLLETECPQYIR (residues 37 to 48), GADVWFK (residues 50 to 56 ), SHEESHKE (residues
86 to 93).
y(6)
4700 MS/MS Precursor 1806.79 Spec #1=>NR(2.00)[BP = 761.3, 361]
0
9.0
389.2
769.4
1149.6
M as s (m /z )
b(14)
b(13)
b(12)
b(11)
y(8)
y(7)
b(5)
y(5)
b(4)
20
y(4)
40
b(3)
b(2)
60
y(9)
b(10)
y(10)
NIETIINTFHQYSVK
80
% Intens ity
% Intensity
100
1529.8
1910.0
Y(10)
Mass (m/z)
4700 MS/MS Precursor 1455.6 Spec #1=>NR(2.00)[BP = 1455.8, 3031]
100
LGHPDTLNQGEFK
0
9.0
315
621
Mass (m/z)
927
Y(11)
b(12)
Y(12)
b(11)
Y(8)
b(9)
b(10)
Y(9)
Y(7)
b(8)
20
b(6)
Y(4)
b(5)
Y(5)
b(3)
40
Y(6)
b(7)
60
% Intensity
% Intensity
80
1233
1539
Mass (m/z)
4700 MS/MS Precursor 1742.68 Spec #1=>NR(2.00)[BP = 1742.7, 562]
100
0
9.0
375.4
741.8
Mass (m/z)
1108.2
Y(13)
b(14)
Y(12)
b(12)
Y(11)
b(7)
Y(8)
b(8)
Y(9)
20
b(6)
Y(7)
b(4)
Y(5)
b(5)
40
b(2)
% Intensity
% Intensity
60
Y(10)
b(10)
VIEHIMEDLDTNADK
80
1474.6
1841.0
Mass (m/z)
Figure S4. MALDI-TOF/TOF spectra with fragment ion signals of peptides obtained from triptic
digestion of S100A9: NIETIINTFHQYSVK (residues 11 to 25), LGHPDTLNQGEFK (residues 26
to 38), VIEHIMEDLDTNADK (residues 58 to 72).
y(1)
4700 MS/MS Precursor 1012.36 Spec #1=>NR(2.00)[BP = 175.1, 644]
WESGYNTR
y(6)
80
0
9.0
221.2
y(5)
b(4)
20
y(3)
y(2)
40
b(2)
60
% Intensity
% Intensity
100
433.4
645.6
Mass (m /z)
857.8
1070.0
Y(9)
Mass (m/z)
4700 MS/MS Precursor 1400.57 Spec #1=>NR(2.00)[BP = 1097.6, 6575]
303.6
b(8)
Mass (m /z)
598.2
Y(10)
Y(8)
Y(6)
b(7)
Y(5)
Y(3)
b(2)
0
9.0
Y(4)
b(5)
20
Y(2)
b(3)
40
Y(1)
60
STDYGIFQINSR
b(6)
80
% Intensity
% Intensity
100
892.8
1187.4
1482.0
Mass (m/z)
TPGAVNACHLSCSALLQDNIADAVACAK
0
9.0
625.8
1242.6
1859.4
Y(20)
Y(19)
b(20)
b(18)
b(17)
Mass (m /z)
Y(17)
Y(16)
b(15)
b(14)
Y(12)
Y(13)
b(9)
Y(6)
20
Y(4)
40
Y(10)
b(10)
b(11)
60
% Intensity
% Intensity
80
b(16)
4700 MS/MS Precursor 2927.46 Spec #1=>NR(2.00)[BP = 2310.7, 84]
100
2476.2
3093.0
Mass (m/z)
Figure S5. MALDI-TOF/TOF spectra with fragment ion signals of peptides obtained from triptic
digestion of Lysozyme C: WESGYNTR (residues 52 to 59), STDYGIFQINSR (residues 69 to 80),
TPGAVNACHLSCSALLQDNIADAVACAK (residues 88 to 115).
3488
Mass (m/z)
3442
3486
3371
20
80
3296
3392
3488
Mass (m/z)
3442
3486
3371
60
20
100
80
3296
3392
3488
Mass (m/z)
3442
3486
3371
3584
PIC
3584
3680
3488
Mass (m/z)
3650
3700
3750
3800
3850
60
NO PIC
40
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T=0 C
20
3650
3700
3750
Mass (m/z)
3800
3584
3680
PIC
40
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T=0 C
20
0
4200
4260
4320
4380
4440
4500
80
4328
60
NO PIC
40
3 months, T=- 80 C
1 month, T=- 80 C
t=0,T=0 C
20
0
4200
4260
4320
4380
Mass (m/z)
4440
4500
100
PIC
60
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
40
20
3650
80
3700
3750
3800
3850
NO PIC
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
40
20
3650
3700
3750
Mass (m/z)
80
4328
60
PIC
40
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
20
0
4200
4260
4320
4380
Mass (m/z)
4440
4500
100
3709
60
0
3600
4328
60
Mass (m/z)
3850
3709
80
0
3600
80
100
3709
80
0
3600
Intensity (A.U.)
0
3600
100
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
3392
20
Mass (m/z)
40
3296
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T=0 C
100
NO PIC
0
3200
40
Mass (m/z)
3680
60
20
PIC
100
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
40
0
3200
3680
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T=0 C
40
0
3200
3584
NO PIC
60
100
Intensity (A.U.)
3392
60
Intensity (A.U.)
Intensity (A.U.)
80
3296
Intensity (A.U.)
20
3709
Intensity (A.U.)
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T=0 C
80
3800
3850
Intensity (A.U.)
40
Intensity (A.U.)
PIC
100
Intensity (A.U.)
3486
60
0
3200
100
100
3442
3371
Intensity (A.U.)
80
Intensity (A.U.)
Intensity (A.U.)
100
80
4328
60
NO PIC
40
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T=0 C
20
0
4200
4260
4320
4380
Mass (m/z)
4440
4500
Figure S6. Examples of peak area variation as a function of different storage conditions in
“extracted” sample groups. Spectra overlays in absolute units highlight the trend of peak area for
m/z= 3371, m/z= 3342, m/z= 3486, m/z= 3709 and m/z= 4328.
60
PIC
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T= 0 C
40
20
4900
4950
5000
Mass (m/z)
5050
NO PIC
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T= 0 C
40
20
4900
4950
5000
Mass (m/z)
5050
Intensity (A.U.)
14691
40
12689
13458
20
11000
12000
13000
14000
PIC
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T= 0 C
15000
13153
80
10835
60
40
12689
20
0
10000
5100
11000
12000
14691
13458
13000
Mass (m/z)
100
14000
NO PIC
3 months, T=- 80 C
1 month, T=- 80 C
t=0, T= 0 C
15000
100
80
4964
60
PIC
3 months, T=- 20 C
1 month, T=- 20 C
t=0,T= 0 C
40
20
4900
4950
5000
Mass (m/z)
5050
13153
80
10835
60
14691
12689
40
20
0
10000
5100
11000
12000
13458
13000
Mass (m/z)
14000
PIC
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T= 0 C
15000
100
100
Intensity (A.U.)
Intensity (A.U.)
Intensity (A.U.)
60
80
4964
60
NO PIC
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T= 0 C
40
20
0
4850
60
100
4964
80
0
4850
10835
Mass (m/z)
100
0
4850
13153
80
0
10000
5100
Intensity (A.U.)
0
4850
Intensity (A.U.)
100
4964
80
4900
4950
5000
Mass (m/z)
5050
5100
Intensity (A.U.)
Intensity (A.U.)
100
13153
80
10835
60
40
12689
13458
14691
20
0
10000
11000
12000
13000
Mass (m/z)
14000
NO PIC
3 months, T=- 20 C
1 month, T=- 20 C
t=0, T= 0 C
15000
Figure S7. Examples of peak area variation as a function of different storage conditions in
“extracted” sample group. Spectra overlays in absolute units highlight the trend of peak area for
m/z= 4964, m/z= 10835, m/z= 12689 m/z= 13153 and m/z= 14691.
13153
100
t=0, T=0 C
% Intensity
80
10835
60
12689
40
4964
20
13458
4328
0
3800
14691
10444
7040
10280
13520
16760
20000
Mass (m/z)
4964
10835
% Intensity
80
4137
20
0
3800
7040
10280
13520
100
4964
10835
80
13458
13780
14005
12689
14691
60
40
13153
t=1 m, T= -20 C
% Intensity
100
16760
60 4137
13458
40
12689
20
20000
0
3800
7040
10835
t=3 m, T= -20 C
13458
4964
13272
60 4137
10444
40
13780
14005
10280
12689
14691
13520
Mass (m/z)
16760
20000
16760
t=3 m, T= -80 C
80
60
40
4137
13272
4964
12689
10444
20
7040
13520
10835
100
20
0
3800
10280
Mass (m/z)
% Intensity
% Intensity
80
14691
10444
Mass (m/z)
100
t=1 m, T= -80 C
13153
20000
0
3800
7040
10280
13458
14691
13520
16760
20000
Mass (m/z)
Figure S8. Examples of MALDI-TOF MS spectra of a GCF control sample immediately processed
(time=0 and T=0°C) and of GCF samples stored on the paper strips and then processed. The
different times and temperatures of storage are shown at top right in each MALDI panel. The
spectra are reported in relative intensity in a m/z range from 3800 to 20000.
100
ALBUMIN
% Intensity
80
60
40
20
0
40000
52000
64000
76000
88000
100000
Mass (m/z)
Figure S9. A typical MALDI-TOF MS spectrum of GCF in the m/z range from 40000 to 100000 is
shown. GCF from one healthy participant to the study was collected with paper strips and eluted
with TFA. MALDI measurements were performed in SA and the spectrum was recorded in linear
mode. The prominent peak of human serum albumin and the absence of any signal derived from the
salivary -amylases (~56 kDa) can be observed.
Table S2. Discriminant peaks in delay extracted samples at different storage conditions
m/z
Protein name
Identification
method
p valuea
p value
p value
p value
-20°C 1M -20°C 3M -80°C 1M -80°C 3M
3371
HNP-2
[Ngo]b
0.03
4.3 10-5
0.07
2.9 10-4
3442
HNP-1
[Ngo]b
8 10-3
3.1 10-5
0.05
5.710-4
3486
HNP-3
[Ngo]b
6 10-3
5.5 10-6
9.5 10-3
8.6 10-5
3709
HNP-4
[Pisano]b
5.4 10-3
2.9 10-5
0.02
3.3 10-3
4137
NI
5.1 10-4
1.9 10-7
9.1 10-3
3.1 10-6
4328
HBD-2
[Mathews]b
6.8 10-5
7.6 10-7
0.01
3.3 10-6
4937
Thymosin 10
[Inzitari]b
4.3 10-4
6.8 10-7
9.6 10-3
8.1 10-5
4964
Thymosin 4
[Inzitari]b
5.9 10-4
6.4 10-5
0.06
4.3 10-3
5793
P-B peptide
[Pisano]b
3.1 10-5
9.4 10-8
8.7 10-4
3.4 10-6
10444
NI
8.8 10-4
9.3 10-6
7.7 10-3
8.8 10-4
10835
S100-A8
3.8 10-6
6.6 10-7
8.1 10-6
4.4 10-6
11367
NI
0.08
8.1 10-3
0.21
8.8 10-3
12689
S100-A9*
1DE-MALDI-TOF/TOF
4.1 10-4
8.7 10-6
4.7 10-3
3.9 10-4
13153
S100-A9
1DE-MALDI-TOF/TOF
9.1 10-5
6.5 10-9
9.3 10-5
9.7 10-6
13458
S100-A9 (glut.)d
1DE-MALDI-TOF/TOF
4.4 10-6
5.1 10-7
8.8 10-4
6.3 10-5
13778
NI
9.4 10-4
3.2 10-6
4.3 10-3
8.2 10-5
14005
NI
0.08
6.6 10-3
0.14
2.8 10-3
14691
Lysozyme C
1.4 10-3
3.9 10-7
0.19
8.9 10-3
1DE-MALDI-TOF/TOF
1DE-MALDI-TOF/TOF
a. The p values were calculated with paired t-test on peak area. Significant p values (less than 0.05) are shown in bold.
b. Reference for protein identification/ extrapolated data from bibliography.
c. NI not identified
d. supposed post-translational modification: glutathionylation
References for protein identification
Ngo, L. H., Veith, P. D., Chen, Y. Y., Chen, D. et al., Mass spectrometric analyses of peptides and
proteins in human gingival crevicular fluid. J. Proteome Res. 2010, 9, 1683-1693.
Pisano, E., Cabras, T., Montaldo, C., Piras, V. et al., Peptides of human gingival crevicular fluid
determined by HPLC-ESI-MS. Eur. J. Oral Sci. 2005, 113, 462-468.
Mathews, M., Jia, H. P., Guthmiller, J. M., Losh, G. et al., Production of beta-defensin
antimicrobial peptides by the oral mucosa and salivary glands. Infect. Immun. 1999, 67, 2740-2745.
Inzitari, R., Cabras, T., Pisano, E., Fanali, C. et al., HPLC-ESI-MS analysis of oral human fluids
reveals that gingival crevicular fluid is the main source of oral thymosins beta(4) and beta(10). J.
Sep. Sci. 2009, 32, 57-63.