Proteomics 2005, 5, 4597–4607
4597
DOI 10.1002/pmic.200401316
REGULAR ARTICLE
MS characterization of multiple forms of alpha-amylase
in human saliva
Christophe Hirtz1, François Chevalier2, Delphine Centeno2,
Valerie Rofidal2, Jean-Christophe Egea1, Michel Rossignol2,
Nicolas Sommerer2 and Dominique Deville de Périère1
1
2
Laboratory of Physiology, UFR d’Odontologie, Université Montpellier 1, Montpellier, France
Laboratory of Proteomics, UR 1199, INRA, Montpellier, France
Alpha-amylase is a major and well-characterized component of human saliva. Recent proteomic
studies suggested that this protein could be observed in more than twenty spots on 2-D gels of
salivary proteins. The aim of this work was to investigate this unexpected redundancy. 2-D gel
electrophoresis was combined with systematic MALDI-TOF MS analysis. More than 140 protein
spots identifying the alpha-amylase were shown to constitute a stable but very complex pattern.
Careful analysis of mass spectra and simultaneous hierarchical clustering of the observed peptides and of the electrophoretic features of spots allowed one to define three major groups. A
main class grouping 90 spots was shown to correspond to full length alpha-amylases that can be
assumed to include isoforms and post-translationally modified forms, a subset of this class being
demonstrated to be N-glycosylated. A second group included short alpha-amylases that are differently truncated in a non-random manner, very likely in the oral cavity. The last class grouped
alpha-amylase forms showing both the N- and C-terminal sequences of the enzyme but displaying a molecular weight that was up to 50% lower than that of the native protein. It is speculated
that the last group of alpha-amylase spots could correspond to proteins submitted to internal
deletions prior to the secretion.
Received: July 16, 2004
Revised: February 15, 2005
Accepted: March 8, 2005
Keywords:
Alpha-amylase / Mass spectrometry / Protein processing / Saliva
1
Introduction
Among body fluids, saliva is particularly easy to collect in
a non invasive way and was proposed as a source of predictive markers for various diseases [1–3]. However, most
techniques used to date in order to characterize the protein composition of saliva, such as SDS-PAGE [4], Western blot [5], ELISA [6] or enzymatic tests [7], did not allow
a large scale investigation. Very recently, three efforts
resulted in the first proteomic characterizations of
saliva by MS [8–10]. Interestingly, in two of these works,
Correspondence: Dr. Christophe Hirtz, Laboratory of Physiology,
UFR d’Odontologie, Université Montpellier 1, Montpellier, France
E-mail: [email protected]
Fax: 133-0-04-67-10-45-89
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
a-amylase, a major protein in saliva, was identified in
multiple protein spots, raising the question of the origin
of this redundancy.
The enzyme a-amylase (EC 3.2.1.1) catalyzes random
hydrolysis of internal alpha 1,4-alpha-glucosidic linkages from
various glucose polymers in saliva and pancreas [11]. Salivary
and pancreatic isoenzymes are the products of two closely
linked genetic loci [12] and each gene, denoted Amy1 for the
salivary and Amy2 for the pancreatic, can occur in a number of
alleles giving rise to at least 12 distinct phenotypes for the salivary isoenzymes and six for the pancreatic [11] forms. At the
protein level, an additional source of diversity is due to post
translational modifications [13], more than 25% of a-amylase
secreted into saliva being assumed to be glycosylated [14–18].
Other reported modifications include post-secretion deamidation of asparagine and glutamine residues [19].
4598
C. Hirtz et al.
With respect to the possible use of salivary a-amylase as a
predictive marker, the goal of this study was to search further
experimental evidences for the multiplicity of a-amylase
spots and to delineate the corresponding subsets. For this
purpose, proteins separated by high resolution 2-D electrophoresis were systematically characterized by MALDI-TOF
MS, and mass spectra corresponding to a-amylase were
carefully inspected and compared.
2
Materials and methods
2.1 Chemicals
Immobiline dry strip gels (180 mm) were from Amersham
Biosciences, the protease inhibitor cocktail from Roche,
modified sequencing grade trypsin from Promega and
C18Zip-Tip from Millipore. Other high purity reagents were
from Sigma or Bio-Rad excepted the kit to measure protein
concentration that was from Pierce.
2.2 Saliva protein extraction
Parafilm-stimulated whole saliva was collected 2 h after usual
breakfast time [20] and complemented with a protease inhibitor cocktail. Saliva samples were centrifuged at 10 000 g
for 15 min, and the supernatant was frozen at 2807C. Protein were precipitated using 90% acetone v/v, 10% TCA v/v
and 0.07% 2-mercaptoethanol v/v. After incubation at 2207C
for 2 h, insoluble material was pelleted at 37 000 g. Pellets
were washed three times with pure acetone containing
2-mercaptoethanol, air dried and solubilised in 9 M urea, 4%
CHAPS w/v, 0.05% Triton X100 v/v and 65 mM DTT. Protein
amount was estimated using the Bradford method [20].
2.3 Enzymatic deglycosylation of salivary protein by
PNGase F
Saliva samples were centrifuged at 10 000 g for 15 min and
the supernatant was mixed with 0.1 M phosphate buffer at
pH 7.5. Samples were heated for 10 min at 1007C in the
presence of 0.02% SDS w/v and 10 mM 2-mercaptoethanol,
prior to 2 h incubation at 377C with PNGase F. Proteins were
analyzed on 11% SDS-polyacrylamide gels and stained with
silver nitrate [21]
2.4 2-DE
Precast IPG strips (pH 3–10 NL) gradient were rehydrated
overnight with protein sample. IEF was carried out using the
IPGphor IEF system (Amersham Biosciences) for a total of
ca. 50 000 Vh. Thereafter, strips were equilibrated for 15 min
in 6 M urea, 50 mM Tris HCl buffer at pH 8.8, 30% glycerol v/
v, 2% SDS w/v and 65 mM DTT, and finally for 15 min in the
same solution excepted that DTT was replaced by 13.5 mM
iodoacetamide. Proteins were finally separated on 12% SDSpolyacrylamide gels, at constant voltage (150 V) and 107C,
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2005, 5, 4597–4607
using an Iso-DALT electrophoresis unit (Amersham Biosciences). Gels were stained with colloidal Coomassie blue.
Gel images were digitalized at 300 dpi with a GS 710 densitometer (Biorad) and analyzed using the Progenesis software
(Non-linear Dynamics). Consistent maps were repeatedly
obtained from different protein samples. For glycoprotein
detection, gels were stained using the Pro-Q Emerald 488
Glycoprotein Gel Kit according to instructions of the manufacturer (http://www.probes.com). Gels were visualized with
a FLA-5000 image analyzer (Fuji) using the 473 nm excitation laser and the long pass filter Y510. Image were acquired
at a 100 mm resolution with a 700 V voltage applied to the
PMT. After glycoprotein detection, gels were stained for total
protein using SYPRO Ruby (http://www.probes.com). Image
was acquired as for Pro-Q Emerald.
2.5 In-gel digestion
Protein spots were excised from gels by hand, and processed
using a Packard Multiprobe II liquid handling robot (Perkin
Elmer). After washing with successively water, 25 mM ammonium bicarbonate, ACN/25 mM ammonium bicarbonate (1:1,
v/v) and ACN, gel fragments were dried at 377C. Protein
digestion was carried out at 377C for 5 h after addition of
0.125 mg trypsin. Resulting fragments were extracted twice
with 50 mL of ACN / water (1:1, v/v) containing 0.1% TFA for
15 min. Pooled supernatants were concentrated to a final volume of ca. 20 mL. Peptides were desalted and concentrated to a
final volume of 3 mL with C18Zip-Tip micro-columns, and
immediately spotted onto the MALDI target by the robot.
2.6 MALDI-TOF MS analysis
Mass spectra were recorded in the reflector mode on a BiFlex
III MALDI-TOF mass spectrometer (Bruker Daltonics).
Automatic annotation of monoisotopic masses was performed using Bruker’s SNAP procedure. The MASCOT
search engine software (Matrix Science), was used to search
the NCBInr database. The following parameters were used
for database searching: mass tolerance of 100 ppm, a minimum of five peptides matching to the protein, carbamidomethylation of cysteine as fixed modification, oxidation of
methionine and pyroglutamylation of glutamine as variable
modifications and one missed cleavage allowed.
3 Results
3.1 Alpha-amylase is present in multiple spots and
constitutes a main component of saliva protein
maps
In order to best characterize the diversity of a-amylase spots
in the saliva proteome, the first analysis was made using gels
covering large pI and MW ranges. More than 350 spots were
detected (Fig. 1). A total of 140 protein spots was identified by
www.proteomics-journal.de
Proteomics 2005, 5, 4597–4607
Clinical Proteomics
4599
Figure 1. 2-D map of human
salivary
proteins.
Proteins
(500 mg) were resolved using pH
3–10NL IPG and 12% SDS-PAGE,
and stained with colloidal Coomassie blue. Alpha-amylase
spots subsequently identified by
MALDI-TOF MS are indicated by
respective spot number.
MALDI-TOF MS with high confidence, owing to both the
criteria selected (see Materials and methods section), the
scores obtained (that were 65% higher in average than the
significance threshold for the queried database), and the
average sequence coverage (that exceeded 40%). These 140
spots identified a total of 26 different proteins in databases,
among which the a-amylase accession (accession number
P04745 in Swiss-Prot database) was the most frequently
matched (67 spots; Table 1). These a-amylase spots differed
largely by both apparent MW on gel (Fig. 1), ranging between
18 and 59 kDa (Table 1), and pI, ranging between 3.5 and 7.6
(Table 1).
3.2 Glycosylation status can not account for all the
multiplicity of Æ-amylase spots
Alpha-amylase was previously assumed to be submitted to
glycosylation [8–10], resulting in the observation of additional isoforms at 59 kDa in addition to the native forms at
56 kDa. Detailed image analysis of this part of gels (Fig. 2, A
left) revealed two series of 45 spots each differing by the pI.
All the twelve spots analyzed in these series identified the
P04745 accession (Table 1). In addition, image analysis
showed that these series accounted for the two thirds of the
total volume of spots identified as a-amylase, the spots at
59 kDa cumulating alone ca. 25% of total a-amylase. In order
to get more insights into the glycosylation status of a-amylase spots, detection of glycosylated proteins was attempted
on 2-D gels using fluorescent dyes. Figure 2 (part B) illustrates the patterns obtained on a same gel with the Pro-Q
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Features of 56 and 59 kDa a-amylase isoforms. (A) left,
analysis of the corresponding 2-D gel area (colloidal Coomassie
blue staining) by the Progenesis software. (A) right, SDS-PAGE
pattern for control and PNGase F-treated samples. (B) multiplexed detection of glycoproteins (left) and total proteins (right)
using Pro-Q Emerald and SYPRO Ruby fluorescent dyes. Spots in
boxes 1–3 were identified by MALDI-TOF MS as a-amylase (box
1), zinc alpha-2 glycoprotein (box 2) and secretory actin binding
protein (box 3).
Emerald dye (left) for the detection of glycoproteins, and with
SYPRO Ruby (right) for total protein detection. Both 56k Da
and 59 kDa a-amylase spots were stained by Pro-Q Emerald
(box 1) whereas none of the 55 a-amylase spots identified
below 56 kDa was detected. Other positive spots were identiwww.proteomics-journal.de
4600
C. Hirtz et al.
Proteomics 2005, 5, 4597–4607
Table 1. Identified a-amylase spots (Swiss-Prot P04745; MW = 55746/pI = 6.34). Sequence coverage (% cov.) is
given as percentage. Observed MW and pI values are deduced from 2-D gels. Clusters for a-amylase spots
refer to the classification of Fig. 4
Spot no Cluster % cov.
Matched
peptides
MW
pI
Spot no Cluster % cov.
Matched
peptides
MW
pI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
18
14
27
18
26
18
21
23
25
27
20
26
12
16
17
19
13
12
24
20
16
17
24
16
16
18
20
19
15
18
11
15
14
19
56
59
56
59
56
59
56
59
56
59
56
59
28
30
29
34
35
37
32
30
30
30
32
33
29
35
35
34
35
37
45
43
46
45
3.5
3.5
5.4
5.4
6.2
6.2
6.6
6.6
6.9
6.9
7.3
7.3
5.8
5.8
6.2
6.2
6.2
6.3
7.0
7.0
7.1
7.0
6.9
6.9
6.9
6.8
6.7
6.6
6.4
6.1
6.0
6.1
6.2
6.2
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
14
12
16
18
20
9
18
22
13
16
10
16
17
18
7
14
18
20
13
18
19
18
11
16
14
18
8
6
7
7
6
6
5
46
47
46
46
46
44
45
45
46
46
37
39
36
32
28
28
43
42
39
34
33
30
29
29
32
28
21
18
21
22
25
23
21
6.1
6.7
6.8
7.0
7.2
7.1
7.3
7.5
7.0
6.9
6.9
7.6
7.5
6.9
6.7
5.9
6.9
7.2
7.7
6.9
6.9
6.6
6.4
6.8
5.9
6.6
6.2
5.7
5.7
5.5
6.4
6.5
5.1
A
A
A
A
A
A
A
A
A
A
A
A
H
H
H
H
B
B
H
H
G
G
H
H
H
H
H
H
B
H
H
B
C
B
42
33
45
28
40
38
51
50
35
40
39
31
27
29
24
30
15
21
56
53
24
23
33
16
14
14
22
30
25
25
26
45
45
51
fied by MALDI-TOF MS as zinc alpha-2 glycoprotein (box 2)
and secretory actin binding protein (box 3), two proteins
known to be glycosylated [22, 23]. In complementary experiments, saliva proteins were treated with PNGase F prior to
electrophoresis. In this case, the band at 59 kDa was almost
undetected after sensitive silver staining (Fig. 2 part A, right).
3.3 PMFs cover a major part of a-amylase sequence
In order to get other insights into the complexity of a-amylase proteome, PMF were compared across the set of spectra.
Only a limited number of peptides was found to be systematically absent from the peptide maps of the 67 spots
(Table 2). A first group corresponded to very short peptides
(with MW lower than 600 Da) and to the long 425G-457K peptide (3595 Da). Other peptides not detected in any spot
included a quite long (3278 Da) and hydrophobic (grand
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C
F
F
F
F
G
G
G
F
F
B
G
G
G
G
G
H
H
H
H
H
H
H
H
H
H
E
E
E
D
D
D
D
45
30
46
52
49
18
47
56
28
36
16
39
43
43
17
34
47
48
40
50
49
44
40
39
31
47
31
19
22
18
19
20
17
average hydropathy score of 0.12) peptide 93I-124R potentially
counter-selected during the ionization process, and a peptide
in the C-terminal part of the protein, 458I-466K (919 Da).
Whereas the C-terminal part of a-amylase was observed
in 60% of spots, the unmodified N-terminal part
(QYSSNTQQGR; MH1 = 1168.53 Da) was not detected in
any digest (Table 2). On another hand, glutamine residues in
position 1 are known to be highly susceptible of pyroglutamylation after tryptic digestion [24], resulting in a loss
of 17 Da. No direct attempt was made here to demonstrate
glutamine pyroglutamylation. However, as a peptide of
1151.53 Da (Fig. 3) was actually observed in 42 alpha-amylase spots (Table 2), it can be hypothesized that the a-amylase
N-terminal peptide was likely modified on the glutamine
residue. Thus 80% of the sequence, including N- and C-terminal peptides appeared to be detectable when comparing
the 67 a-amylase spots (Fig. 4, panel A).
www.proteomics-journal.de
Clinical Proteomics
Proteomics 2005, 5, 4597–4607
4601
Table 2. Potential a-amylase peptides. Only technically detectable peptides, with m/z ranging between 500 and
3500 Da are shown (one missed cleavage, MC, allowed). Mass data correspond to unmodified peptides,
excepted for peptide 1* (pyroglutamylation of 1Gln). Modifications allowed for database query included
carbamidomethylation of Cys (fixed modification) and Met oxidation/Gln pyroglutamylation (variable
modifications). Occurrence, number of spots where the peptide was observed (none of these peptides
could be observed in control gel pieces lacking detectable protein material)
Peptide No
Position m/z
Peptide sequence
MC
Occurrence
1
1*
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
20
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
41
43
44
45
46
47
48
49
50
51
52
53
54
55
56
01– 10
01– 10
01– 20
11– 20
11– 30
21– 30
21– 35
31– 35
31– 61
36– 61
62– 68
62– 72
69– 80
73– 80
73– 85
81– 85
81– 92
86– 92
93–124
125–140
125–142
141–158
143–158
143–161
159–172
162–172
162–176
173–176
173–178
177–195
179–195
179–200
196–200
196–208
201–208
201–213
209–213
214–243
244–252
244–257
253–257
253–261
258–267
262–267
262–268
268–273
269–273
269–278
274–278
274–291
279–291
279–303
QYSSNTQQGR
QYSSNTQQGR
QYSSNTQQGRTSIVHLFEWR
TSIVHLFEWR
TSIVHLFEWRWVDIALECER
WVDIALECER
WVDIALECERYLAPK
YLAPK
YLAPKGFGGVQVSPPNENVAIHNPFRPWWER
GFGGVQVSPPNENVAIHNPFRPWWER
YQPVSYK
YQPVSYKLCTR
LCTRSGNEDEFR
SGNEDEFR
SGNEDEFRNMVTR
NMVTR
NMVTRCNNVGVR
CNNVGVR
IYVDAVINHMCGNAVSAGTSSTCGSYFNPGSR
DFPAVPYSGWDFNDGK
DFPAVPYSGWDFNDGKCK
CKTGSGDIENYNDATQVR
TGSGDIENYNDATQVR
TGSGDIENYNDATQVRDCR
DCRLSGLLDLALGK
LSGLLDLALGK
LSGLLDLALGKDYVR
DYVR
DYVRSK
SKIAEYMNHLIDIGVAGFR
IAEYMNHLIDIGVAGFR
IAEYMNHLIDIGVAGFRIDASK
IDASK
IDASKHMWPGDIK
HMWPGDIK
HMWPGDIKAILDK
AILDK
LHNLNSNWFPEGSKPFIYQEVIDLGGEPIK
SSDYFGNGR
SSDYFGNGRVTEFK
VTEFK
VTEFKYGAK
YGAKLGTVIR
LGTVIR
LGTVIRK
KWNGEK
WNGEK
WNGEKMSYLK
MSYLK
MSYLKNWGEGWGFMPSDR
NWGEGWGFMPSDR
NWGEGWGFMPSDRALVFVDNHDNQR
0
0
1
0
1
0
1
0
1
0
0
1
1
0
1
0
1
0
0
0
1
1
0
1
1
0
1
0
1
1
0
1
0
1
0
1
0
0
0
1
0
1
1
0
1
1
0
1
0
1
0
1
0
42
0
54
0
33
0
2
0
29
31
9
0
58
56
0
0
1
0
45
0
0
50
0
0
44
23
0
2
0
45
0
0
0
25
0
1
13
51
0
0
1
0
1
0
0
7
0
1
0
44
0
1168.53
1151.53
2437.20
1287.68
2502.26
1233.59
1805.93
591.35
3562.81
2990.48
884.45
1357.69
1426.64
953.40
1554.70
620.32
1362.67
761.37
3278.47
1814.80
2045.91
1970.89
1739.78
2113.92
1473.81
1099.67
1632.93
552.28
767.40
2134.11
1918.98
2433.26
533.29
1497.75
983.48
1523.80
559.35
3441.75
1002.43
1606.75
623.34
1042.56
1077.64
658.42
786.52
761.39
633.30
1255.61
641.33
2160.96
1538.65
2947.33
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.proteomics-journal.de
4602
C. Hirtz et al.
Proteomics 2005, 5, 4597–4607
Table 2. Continued
Peptide No
Position m/z
Peptide sequence
MC
Occurrence
57
58
59
60
61
62
63
64
65
66
67
68
69
71
72
73
74
75
76
77
78
80
82
83
84
85
86
87
292–303
292–319
304–319
304–322
320–337
323–337
323–343
338–343
338–346
344–352
347–352
347–368
353–368
369–387
369–389
388–392
390–398
393–398
393–421
399–421
399–424
425–457
458–466
458–474
467–474
467–495
475–495
475–496
ALVFVDNHDNQR
ALVFVDNHDNQRGHGAGGASILTFWDAR
GHGAGGASILTFWDAR
GHGAGGASILTFWDARLYK
LYKMAVGFMLAHPYGFTR
MAVGFMLAHPYGFTR
MAVGFMLAHPYGFTRVMSSYR
VMSSYR
VMSSYRWPR
WPRYFENGK
YFENGK
YFENGKDVNDWVGPPNDNGVTK
DVNDWVGPPNDNGVTK
EVTINPDTTCGNDWVCEHR
EVTINPDTTCGNDWVCEHRWR
WRQIR
QIRNMVNFR
NMVNFR
NMVNFRNVVDGQPFTNWYDNGSNQVAFGR
NVVDGQPFTNWYDNGSNQVAFGR
NVVDGQPFTNWYDNGSNQVAFGRGNR
GFIVFNNDDWTFSLTLQTGLPAGTYCDVISGDK
INGNCTGIK
INGNCTGIKIYVSDDGK
IYVSDDGK
IYVSDDGKAHFSISNSAEDPFIAIHAESK
AHFSISNSAEDPFIAIHAESK
AHFSISNSAEDPFIAIHAESKL
0
1
0
1
1
0
1
0
1
1
0
1
0
0
1
1
1
0
1
0
1
0
0
1
0
1
0
1
59
0
60
0
0
39
10
0
10
0
28
0
35
16
0
1
0
15
0
54
0
0
0
0
29
0
40
19
1427.70
3024.48
1615.80
2020.04
2102.07
1697.83
2421.17
742.36
1181.59
1196.58
757.35
2465.14
1726.80
2188.94
2531.12
758.44
1177.63
780.38
3346.54
2585.18
2912.35
3594.71
919.47
1796.88
896.44
3148.52
2271.10
2384.19
Figure 3. PMF for spot 19. Features of peptides are detailed in
Table 2; t, autolysis peaks from
trypsine; 1*, peptide 1 with pyroglutamylated 1Gln; 15*, peptide 15 with oxidated 82Met; 33*,
peptide 33 with oxidated 183Met.
3.4 Saliva contains different clusters of Æ-amylase
spots
Alpha-amylase spots differed mainly by both their apparent
MW on gel (Fig. 1), ranging between 18 and 59 kDa (Table 1),
and the sequence regions covered by the PMFs (Fig. 4, panel
C). In order to compare simultaneously these features
throughout the spots, a distance matrix was established by
computing Euclidean distances. For this calculation, two
kinds of data were simultaneously taken into account: (i) the
observation of at least one peptide in the 50 first amino acids
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of the N-terminal region (1Q-50A), in the 100 aminoacids of
the central part (201H-301N) or in the 50 last amino acids of the
C-terminal region (447G-496L), and (ii) the MW of the spot on
gels (below 25 kDa, between 25 kDa and 45 kDa, between
45 kDa and 55 kDa, and above 55 kDa). The rational for peptide selection was that each of the selected part of the
sequence simultaneously is informative about possible
important alterations and corresponds to a sufficiently large
number of peptides (respectively 9, 7 and 21 peptides) to
minimize the risk that slight variations in mass spectra
observed between replicates from different gels could influwww.proteomics-journal.de
Proteomics 2005, 5, 4597–4607
Clinical Proteomics
4603
Figure 4. Mass features of aamylase identified spots. (A)
Coverage of the a-amylase
sequence by the total population of peptides identified (black
boxes) in the different ,-amylase spots. (B) Simultaneous
clustering of the 67 a-amylase
spots according to (i) the MW
range measured on gels and (ii)
the MS identification of peptides
in the N-terminal, C-terminal
and central regions of the
sequence. (C) Individual spot
coverage of the a-amylase
sequence by peptides identified
(black boxes) by MALDI-TOF
MS.
ence further classification. The distances where then compared by hierarchical clustering using the Ward’s criterion.
Accordingly, the structure of a-amylase-related data appeared
to be characterized by the presence of very homogeneous
clusters of similar spots, occasionally agglomerated with one
somehow more distant spot (Fig. 4, panel B).
A first cluster (cluster A; Fig. 4, panel B) of very similar
spots grouped exactly the 12 spots that had been picked along
the two series detected at 56 and 59 kDa (Fig. 2). These spots
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
were identified with high sequence coverage (ca. 50%) and
the matched peptides included both the N- and C-terminal
regions.
Six clusters (clusters B to G; Fig. 4, panel B) grouped
spots all lacking a part of one of the terminal sequences and
differing by MW. The differences between these clusters
relied on the length of the portion not detected, accounting
for either one eighth or one third of the sequence, and on its
position, either on the N- or C-terminal part.
www.proteomics-journal.de
4604
C. Hirtz et al.
Proteomics 2005, 5, 4597–4607
Finally, another large cluster (cluster H; Fig. 4, panel B)
corresponded to proteins showing PMFs covering all the
sequence regions, including the N- and C-terminal parts, but
with low MW ranging between 28 and 43 kDa. In addition,
these proteins showed ca. 80% of the peptides found in the
spots at 56 kDa from cluster A.
In order to get more insights into this situation, the MW
observed on 2-D gels was compared to the cumulated mass
of all peptides identified for each protein in this cluster
(Fig. 5). Whereas for most spots the mass observed on the gel
was higher than that calculated from the PMFs, for some of
them the sum of the masses of all peptides detected was
found to reach the MW values measured on gels.
3.5 Reliability and stability of the classification
Protein maps similar to that shown in Fig. 1 were repeatedly
obtained with saliva protein samples from healthy donors
differing by age and gender. However, for consistency, MS
results presented above corresponded to a same saliva sample where MS characterization had been performed twice. In
order to assess the reliability of the classification, another
protein map from a different healthy donor was matched by
image analysis, and the 67 spots corresponding to those previously identified as a-amylase (Fig. 1) were analyzed by
MALDI-TOF MS. Sixty five spots were unambiguously characterized as a-amylase (P04745 accession), but no result
could be obtained for two spots of low abundance. Detailed
inspection of PMFs showed that, although not exactly all the
same peptides could be detected for each pair of matched
spots, the same classes of a-amylases were still observed with
respect to protein MW on gel and to the detection of at least
one peptide in the three selected regions (Fig. 6). This con-
Figure 6. Biological variability of a-amylase classification. Sixty
five spots from donor 2 were matched to the gel from Fig. 1
(donor 1) and confirmed as a-amylase by MALDI-TOF MS. 56/
59 kDa amylases, spots showing MW above 55 kDa and peptides
in both N-terminal, C-terminal and central part of the sequence
(Fig. 4, cluster A); N-ter deletion, spots showing no peptide in the
N-terminal region (Fig. 4, clusters B-D); C-ter deletion, spots
showing no peptide in the C-terminal region (Fig. 4, clusters E-G);
low MW amylases, spots showing peptides in both N-terminal, Cterminal and central part of the sequence, but MW below 55 kDa
(Fig. 4, cluster H).
cerned as well spots at 56/59 kDa showing both terminal
sequences, spots lacking one of the terminal sequences and
differing by MW, and spots showing peptides covering all the
sequence regions but low MW (respectively, cluster A, clusters B to G and cluster H, Fig. 4).
4
Discussion
Detailed analysis of PMFs shows that previously overlooked
features concerning a-amylase can be evidenced by MALDITOF MS. For instance, whereas all the 70 PMFs supporting
the identification of a-amylase in the three independent proteomic characterizations of saliva published to date failed to
detect the N-terminal sequence [8–10], this sequence was
observed here in the two-thirds of a-amylase spots when taking into account a well-known glutamine modification. On
another hand, our results suggest that the a-amylase proteome displays an unexpected complexity. For task of simplicity, this diversity is categorized below into three main situations: isobaric a-amylases differing by the pI, truncated lower
MW a-amylases and alternative a-amylase forms.
4.1 Isobaric alpha-amylases differing by the pI
Figure 5. Relationship between MW measured on gel and the
cumulated mass of peptides identified by MALDI-TOF MS for
spots from cluster H.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Series of spots differing by the pI are observed at 56 and
59 kDa (Fig. 4, cluster A) and the 12 spots analyzed in these
series showed identical features in different samples (Fig. 6).
www.proteomics-journal.de
Proteomics 2005, 5, 4597–4607
Very similar patterns were also present on gels supporting
previous investigations of saliva [8–10], indicating that they
constitute a signature of the proteome of this fluid. At least
three hypotheses can be raised to account for this situation.
First, according to available data, the presence of isoforms
and/or allelic forms can be expected [25]. This is likely for the
set of spots observed at 56 kDa, the MW of native a-amylase.
On the basis of previous data, PTMs constitute another possible cause. Especially, glycosylation could be taken as
responsible for the spot line at 59 kDa, following the explanation proposed in recent papers [8–10]. Actually, this interpretation relies on conclusions derived from pioneer characterization of saliva proteins by gel filtration [14] that were reused, during three decades but with no additional experimental evidence, in works resolving saliva proteins by 1-D [4]
and then 2-D electrophoresis [8–10].
In the present work, no conclusion can be derived for
56 kDa a-amylases that are unaffected by PNGase F but
stained by Pro-Q Emerald dye. As the protein is observed at
its native molecular weight, the occurrence of non-specific
staining can not be ruled out although it should be noticed
that the other proteins stained on the gel are known to be
glycosylated. By contrast, combined use of the glycoprotein
detection stain and PNGase F treatment strongly suggests
the presence of N-linked chains on 59 kDa a-amylases. On
another hand, it should be pointed out that one of the two
potential N-glycosylation sites of a-amylase (412N) was
detected in all the analyzed spots. This would suggest that Nglycosylation of 59 kDa spots would occur only on the other
potential site (461N) that systematically failed to be detected.
Additional modifications could also contribute to the
multiplicity of spots according to pI within each of the two
series. Owing to the number of Asn and Gln residues in
P04745 sequence (respectively 41 and 12), protein deamidation may be involved [19, 26]. Alternatively, the occurrence of
conformational isomers was recently proposed as a mechanism contributing to the multiplication of spots at the same
MW [27]. In the absence of complementary data to evaluate
their relative contribution, all the three mechanisms can be
hypothesized to participate to the occurrence of isobaric
a-amylase forms. Interestingly, it should be noticed that a
similar situation was recently described in plants, for a-amylase from germinating barley seeds [28], raising the hypothesis that such feature could be common to this enzyme in
various organisms and tissues. In this view, our results
extend previous reports and emphasize that as many as 90
spots could participate to this main subset of a-amylases.
4.2 Truncated lower MW Æ-amylases
Alpha-amylase spots showing an apparent MW lower than
that of the native protein were recently observed in saliva
2-D gels and hypothesized to be generated by degradations
[9]. In the present work, 31 truncated a-amylases constitute
different clusters according to the extent of the truncation
and that terminal part which is affected (Fig. 4, clusters B to
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Clinical Proteomics
4605
G). For instance, clusters B and C correspond to a-amylases
lacking ca. 60 amino acids in N-terminal part of the
sequence, whereas clusters F and G show a loss of ca. 75
amino acids in C-terminal part. By opposition, for clusters D
and E, the lacking sequences concern ca. 300 amino acids.
Such classification relies on the capacity of MALDI-TOF MS
and previous protein treatment steps to provide reliable
PMFs. In this view, it should be emphasized that the same
types of classes can be demonstrated from different samples
(Fig. 6), with difference only in their size. This situation can
be assumed to reveal as well the usual experimental variation in PMFs obtained for spots matched in 2-D gels from
different donors as biological variation between donors. On
another hand, although the precise border of deletions
remains unknown, due to the uneven distribution of acidic
and basic residues along the sequence, the shifts in pI (and
MW) observed on gel are consistent with changes associated
to truncations (calculated using the “Compute pI/Mw”
ExPASy tool http://www.expasy.org/tools/pi_tool.html). For
instance, with a pI predicted as to 6.3 for native a-amylase,
the deletions of 60 N-terminal amino acids or 75 C-terminal
amino acids (peptides 1Q-61R or 422G-496L) are calculated to
lead to pI values of respectively 6.2 and 7.2, whereas the
average pI amounts to 6.2 for spots from clusters B and C,
and to 7.0 for spots from clusters F and G. On another hand,
truncated forms are observed on a narrower pI range than
56 kDa a-amylases that cover nearly 4 pH units. As long as
deamidation can be taken as responsible for the multiplicity
of spots, the extent of the pI range should be related to the
cumulated Asn and Gln content, and estimation of this
content can be obtained from observed peptides. Although
possibly underestimated, these calculations are consistent
with the pI ranges observed. For instance, whereas fulllength amylases cumulate 53 Asn and Gln, this content falls
to 40 for spots from clusters F and G that cover a 1.7 pH
units pI range, and to 22 for spots from clusters B and C that
cover a two times narrower pI range. Therefore, collectively,
our data strongly support the hypothesis of truncations,
raising the question of their origin. It is likely noteworthy
that spots from these clusters do not define a continuum of
deletions and that no spot corresponding to only internal
fragments was observed. On another hand, these spots
define patterns that can be evidenced with saliva from different donors, and are not affected by omission of protease
inhibitors during the collect and storage, nor by prolonged
storage, even at room temperature. Therefore, this indicates
that degradations should occur in the oral cavity, despite the
natural abundance of protease inhibitors in saliva like
cystatins [10]. Furthermore, our data suggest that these
degradations would involve very specific processes, affecting
only one terminal sequence, to different but limited extents,
and in a way excluding degradation of the other terminal
sequence. In the case of clusters D and E, however, the possibility of a combination of initial cleavage by endoproteases,
followed by truncation by exoproteases and resulting in the
two clusters, can not be excluded. Collectively, all these difwww.proteomics-journal.de
4606
C. Hirtz et al.
ferent features argue for non-generalized processing of
a-amylases in saliva, resulting in the constitutive presence of
numerous truncated forms.
4.3 Alternative Æ-amylase forms
Perhaps the most intriguing situation corresponds to cluster H
(Fig. 4), which constitutes a large and stable cluster (Fig. 6).
These spots possess peptides in both the N- and C-terminal
regions, but display MW on gel ranging between 28 and
45 kDa, and cover a pI range between 5.8 and 7.7. As these
alternative forms do not display isobaric electrophoretic profile,
the occurrence of isoforms or alleles, and that of deamidation
events, are likely not responsible for the diversity of spots in this
cluster. In addition, for some of them, the cumulated mass of
observed peptides reaches the MW estimated from gels, thus
suggesting that most of the actual sequence is detected in the
fingerprints and, conversely, that at least part of undetected
peptides could be really absent in the protein. Several hypothesis can be envisaged to account for this unexpected situation.
A first explanation resides in abnormal electrophoretic
migrations. Such cases correspond most frequently to
restricted migrations, thus simulating an increased MW, in
opposition with the present situation. On another hand,
normal migrations are observed here for all other a-amylase
spots, including native proteins at 56 kDa, heavier proteins at
59 kDa and smaller truncated proteins. Therefore, an overmigration specific to spots from cluster H seems rather
unlikely and alternative explanations are to be researched.
The involvement of degradations constitutes a second
hypothesis, provided that they allow for the simultaneous
presence of N- and C-terminal peptides in the spots. Two
kinds of processes would be able to produce such a situation.
Firstly, this could occur through the cleavage of the protein
into peptides and their subsequent association by disulfide
bonds in the case of incomplete reduction and alkylation.
Actually, cysteine residues are present in all but the central
(209A-273K) regions of a-amylase sequence, making possible
the linkage of terminal parts. However, peptides from the
central region are also observed in all spots from cluster H,
excepted one (spot no 57), making it unlikely to observe spots
with MW as low as 28 kDa. Alternatively, the simultaneous
observation of both terminal parts could result from comigration of oppositely truncated forms showing the same
MW. For instance, deletion of half the sequence would produce an N-terminal and a C-terminal polypeptide of 28 kDa
each, the MW of spot no 60 (Fig. 5), whereas a deletion of 280
residues would result into two polypeptides of 32 kDa each,
the MW of spots no 19 and 23 (Fig. 5). However, in both
cases, the resulting polypeptides would display very large
differences in pI, in excess of 1.3 pH unit, precluding their
co-migration. Therefore, this hypothesis can not account for
the observation of spots with close pI.
A last hypothesis is the occurrence of internal deletions.
Protein splicing processes in which an intein fragment
removes itself from the precursor protein that religates fur 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2005, 5, 4597–4607
ther its two flanks are known, including in Eukaryotes [29].
Database search with the complete a-amylase sequence
(http://www.neb.com/inteins/int_id.html) shows the presence of intein-like motifs in the two longest regions (31 and
46 amino acids) not observed in cluster H spots. However, no
complete signature can be detected and the sequences not
observed are more than three-times shorter than in the
shortest inteins previously described. Therefore, according to
available data, it seems unlikely that internal deletions in
a-amylase could arise from intein-mediated auto-processing.
On another hand, very recently, the excision of a 40 amino
acid segment followed by protein splicing was demonstrated
in human cells [30]. This was further confirmed for the
deletion of another tetrapeptide followed by peptide splicing
[31]. In both cases, the process involved the proteasome and a
model was proposed suggesting that splicing could occur
with any fragment produced by the proteasome [31]. In the
absence of complementary data, however, it can be only
speculated that similar mechanisms could be involved here.
In this case, the proteins from cluster H would be produced
prior to secretion and not processed in the oral cavity.
4.4 Possible functional significance implications of
saliva complexity
Above data indicate that various mechanisms participate to
the observed diversity of a-amylase patterns. As several of
them do not rely on simple protein degradation, this points
to the significance of this situation. Resolution of the structure of a-amylase previously identified those residues critical
for the catalytic activity [32]. Interestingly, excepted for truncated proteins, all these residues are located in peptides
observed in all the spots, leaving the possibility that these
proteins could maintain hydrolysis activity. Furthermore, for
cluster H spots, in silico structure modeling (http://www.
expasy.org/swissmod/SWISS-MODEL.html), using the sequence obtained by concatenating only the observed peptides, still gives best scores for a-amylase structure. Therefore, it is tempting to speculate that at least a part of these
a-amylase spots could correspond to functional proteins,
possibly differing by their substrate specificity and/or specific activity, as recently shown for a mutated a-amylase
lacking a glycine-rich loop [33].
In conclusion, systematic analysis of a-amylase spots by
high resolution 2-D gel and MALDI-TOF MS reveals a large
redundancy defining a complex but stable pattern. When
taking also into account all spots detected by image analysis
in the 56 and 59 kDa series, a total of more than 140 a-amylase spots is thus observed. These identify different subsets.
Beside a major class that is far larger than previously detected and corresponds likely to native and post-translationally
modified isoforms, two smaller subsets accounting for ca.
20% of spots each are delineated. One of them is proposed to
result from non-random truncation occurring in the oral
cavity, whereas the second one includes proteins showing
internal deletion that are assumed to be produced prior to the
www.proteomics-journal.de
Proteomics 2005, 5, 4597–4607
secretion. The extent of this diversity is likely to open new
perspectives and challenges for the use of a-amylase in noninvasive diagnostic.
This work was supported by I.F.R.O grants and benefited of
the support of the proteomics platform from the Montpellier Languedoc-Roussillon Genopole. Authors thanks Dr N Chapal and
Dr L Molina for initial help in the project.
Clinical Proteomics
4607
Genetics and Evolution. 1975 Vol 4, p. 745, Academic press,
New York.
[14] Kauffman, D. L., Watanabe, S., Evans, J. R., Keller, P. J.,
Archs. Oral. Biol. 1973, 18, 1105–1111.
[15] Bank, R. A., Hettema, E. H., Arwert, F., Amerongen, A. V.,
Pronk, J. C., Electrophoresis 1991, 12, 74–79.
[16] Ragunath, C., Sundar, K., Ramasubbu, N., Protein Expr.
Purif. 2002, 24, 202–211.
[17] Bank, R. A., Hettema, E. H., Muijs, M. A., Pals, G., et al.,
Electrophoresis 1991, 12, 1032–1041.
[18] Kandra, L., Gyemant, G., Carbohydr. Res. 2000, 329, 579–
585.
5
References
[1] Markopoulos, A. K., Belazi, M. A., Drakoulakos, D., Diabetes
Res. Clin. Pract. 1997, 38, 169–172.
[2] Davidson, G. P., Koheil, A., Forstner, G. G., Pediatr. Res. 1978,
12, 967–970.
[3] Chen, Y. C., Li, T. Y., Tsai, M. F., Rapid Commun. Mass Spectrom. 2002, 16, 364–369.
[19] Karn, R. C., Shulkin, J. D., Merritt, A. D., Newell, R. C., Biochem. Genet. 1973, 10, 341–350.
[20] Bradford, M., Anal. Biochem. 1976, 72, 248–254.
[21] Mortz, E., Krogh, T. N., Vorum, H., Gorg, A., Proteomics 2001,
1, 1359–1363.
[22] Araki, T., Gejyo, F., Takagaki, K., Haupt, H., et al., Proc. Natl.
Acad. Sci. USA 1988, 85, 679–683.
[4] Beeley, J. A., Sweeney, D., Lindsay, J. C., Buchanan, M. L., et
al., Electrophoresis 1991, 12, 1032–1041.
[23] Schenkels, L. C., Schaller; J., Walgreen-Weterings, E., Schadee-Eestermans, I. L., et al., Biol. Chem. Hoppe-Seyler. 1994,
375, 609–615.
[5] Fabian, T. K., Gaspar, J., Fejerdy, L., Kaan, B., et al., Med. Sci.
Monit. 2003, 9, 62–65.
[24] Thiede, B., Lamer, S., Mattow, J., Siejak, F., et al., Rapid
Commun. Mass Spectrom. 2000, 14, 496–502.
[6] Todd, A. L., Ng, W. Y., Lee, Y. S., Loke, K. Y., Thai, A. C., Diabetes Res. Clin. Pract. 2002, 57, 171–177.
[25] Bank, R. A., Hettema, E. H., Muijs, M. A., Pals, G., Hum.
Genet. 1992, 89, 213–222.
[7] Musumeci, V., Cherubini, P., Zuppi, C., Zappacosta, B., et al.,
J. Oral. Pathol. Med. 1993, 22, 76.
[26] Sarioglu, H., Lottspeich, F., Walk, T., Jung, G., Eckershorn, C.,
Electrophoresis 2000, 21, 2209–2218.
[8] Yao, Y., Berg, E. A., Costello, C. E., Troxler, R. F., Oppenheim,
F. G., J. Biol. Chem. 2003, 278, 5300–5308.
[27] Berven, F. S., Karlsen, O. A., Murrell, J. C., Jensen, H. B.,
Electrophoresis 2003, 24, 757–761.
[9] Ghafouri, B., Tagesson, C., Lindahl, M., Proteomics 2003, 3,
1003–1010.
[28] Ostergaard, O., Melchior, S., Roepstorff, P., Svensson, B.,
Proteomics 2002, 2, 733–739.
[10] Vitorino, R., Lobo, M., J. C., Ferrer-Correira, A., J.,, Dubin, J.
R., Proteomics 2004, 4, 1109–1115.
[11] Merrit, A. D., Karn, R. C., The Human a-amylases, 1977, Plenum Press. New York.
[12] Merrit, A. D, Lovrien, E. W., Rivas, M. L., Conneally, P. M.,
Am. J. Hum. Gen. 1973, 25, 523.
[13] Karn, R. C., Schulkin, J. D., Meritt, A. D., Newell, R. C.,
Genetic and Post-translational Mechanisms Determining
Human Amylase Isoenzyme Heterogeneity. Isozymes.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[29] Paulus, H., Annu. Rev. Biochem. 2000, 69, 447–496.
[30] Hanada, K. I., Yewdell, J. W., Yang, J. C., Nature 2004, 427,
252–256.
[31] Vigneron, N., Stroobant, V., Chapiro, J., Ooms, A., et al.,
Science 2004, 304, 587–590.
[32] Ramasubbu, N., Paloth, V., Luo, Y., Brayer, G. D., Levine, M.
J., Acta Cryst. D 1996, 52, 435–446.
[33] Ramasubbu, N., Ragunath, C., Mishra, P. J., J. Mol. Biol.
2003, 325, 1061–1076.
www.proteomics-journal.de