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1
Simple quantitation for potential serum disease biomarker 1
peptides, primarily identified by a peptidomics approach in the 2
serum with hypertensive disorders of pregnancy 3
4
Kensuke Hamamura1,2, Daisuke Nonaka3, Hitoshi Ishikawa4, Michio Banzai5, Mitsuaki 5
Yanagida1, Michio Nojima2, Koyo Yoshida2, Lyang-Ja Lee3, Kenji Tanaka3, Kenji 6
Takamori1, Satoru Takeda2 and Yoshihiko Araki1,2 7
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1Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of 9
Medicine, Chiba, Japan; 2Department of Obstetrics and Gynecology, Juntendo University Graduate 10
School of Medicine, Tokyo, Japan; 3Membrane Protein and Ligand Analysis Center, Protosera Inc., 11
Hyogo, Japan; 4Department of Health Information Management, Yamagata Saisei Hospital, Yamagata, 12
Japan; 5Department of Obstetrics and Gynecology, Yamagata Saisei Hospital, Yamagata, Japan 13
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Corresponding author: Yoshihiko Araki, Institute for Environmental and 15
Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 16
Tomioka, Urayasu, Chiba 279-0021, Japan 17
Email: [email protected] 18
19
DECLARATIONS 20
Competing interests: None declared by all authors. 21
Funding: This study was funded in part by Grants-in-Aid for General Scientific 22
Research/‘High-Tech Research Center’ Project for Private Universities: matching fund 23
subsidy from the Ministry of Education, Culture, Sports, Science & Technology, Japan; 24
2
a grant (A-STEP) from Japan Science and Technology Agency; and Juntendo University 25
Young Investigator Joint Project Award. 26
Ethical approval: The Ethic Committee board members of Juntendo University and 27
Yamagata Saisei Hospital approved this study (registration #19-017 and #144, 28
respectively). 29
Guarantor:YA 30
Contributorship: KH, DN, MY, KTan, KTak, ST and YA researched the literature 31
conceived the study, managed grant application and data analysis; KH, DN, LJL, 32
collected experimental data; HI, MB, MN and KY collected samples, analyzed clinical 33
data, and reviewed the statistical protocol; MY and YA contributed to design the study 34
protocol; KH and YA wrote the manuscript. All authors reviewed and edited the 35
manuscript and approved the final version of the manuscript. 36
Acknowledgements: The authors are indebted to clinical staff of Yamagata Saisei 37
Hospital and Juntendo Urayasu Hospital for their assistance. We gratefully 38
acknowledge Drs. Kyoichi Asada (Protosera Inc.) and Hiroshi Yoshitake (Juntendo 39
University) for their helpful discussion, technical supports, and encouragement 40
throughout the course of this study. 41
42
43
3
Abstract 44
45
Background: We previously reported peptide candidates of disease biomarkers 46
(DBMs) for pregnancy-induced hypertension syndrome (PIH) using a novel peptidomic 47
analytical method, BLOTCHIP®-MS. The aim of this study was to establish a 48
sandwich enzyme-linked immunosorbent assays (ELISA) system for quantitation of 49
such peptides and to validate their usefulness as DBMs of PIH including gestational 50
hypertension/preeclampsia (GH/PE). 51
Methods: We focused on three peptide fragments, kininogen-1438-456 (PDA039), 52
kininogen-1439-456 (PDA044) and cysteinyl α2-HS-glycoprotein341-367 (PDA071). 53
Using polyclonal antibodies (pAbs) specific for each peptide, suitable conditions for the 54
sandwich ELISA system were investigated. The quantitative ELISA values were 55
confirmed by quantitative matrix assisted laser desorption/ionization time-of-flight MS 56
analyses. Using the established ELISA systems, sera from GH/PE patients and paired 57
serum samples from healthy pregnant females were analyzed. 58
Results: The optimum sandwich ELISA conditions for PDA039/044 quantitation were 59
developed. Quantitation of PDA071 by ELISA failed, presumably due to issues with 60
pAb specificity for the native peptide. Bland-Altman plots showed a satisfactory 61
correlation between the serum PDA039/044 concentration by ELISA and that by 62
quantitative MS analysis. Although the PDA044 level showed no significant change 63
during pregnancy, including in GH/PE patients, the serum PDA039 level was 64
significantly increased (P < 0.0001) in the patients. 65
Conclusions: The simple quantitation technology for PDA039 by ELISA was 66
established for the first time. PDA039 is confirmed its clinical utility as a DBM for 67
4
GH/PE by the ELISA system using clinical samples. The information provided from 68
present study would be new valuable addition in the field of GH/PE research. 69
70
Keywords: Sandwich enzyme-linked immunosorbent assays, Peptidomics, 71
Hypertensive disorders of pregnancy, Serum biomarkers72
5
Introduction 73
74
Hypertensive disorders of pregnancy such as pregnancy-induced hypertension 75
syndrome (PIH) is one of the most serious complications of pregnancy.1 The 76
continuous high blood pressure of these patients sometimes causes a convulsive state 77
known as eclampsia,2 a leading cause of substantial maternal/fetal morbidity and 78
mortality.3 Although the pathological relationship between gestational hypertension 79
(GH) and preeclampsia (PE) is still controversial among the hypertensive disorders of 80
pregnancy,1,4 the Japan Society for the Study of Hypertension in Pregnancy provides the 81
clinical classification of PIH that includes both GH and PE.5 At present, the most 82
effective method of predicting the syndrome is monitoring of blood pressure despite the 83
performance of numerous basic and clinical studies. However, no essential treatment 84
method for GH/PE has been developed. Instead, patients must hope for spontaneous 85
recovery after delivery. Thus, new disease biomarkers (DBMs) for GH/PE should be 86
developed to improve clinical management and diagnostic prediction. Accurate 87
monitoring using such DBMs may reduce fetal and maternal mortality due to GH/PE. 88
To identify novel DBMs for intractable ‘poor-prognosis’ diseases such as GH/PE, 89
comprehensive proteomic analysis of humoral fluids is one of the most promising 90
experimental approaches.6 Because more than 20,000 peptide fragments derived from 91
various precursor proteins circulates in peripheral blood,7 we are currently focused on 92
proteomic (peptidomic) analyses that have potential to facilitate identification of novel 93
DBM(s) in blood samples.8-10 However, DBM discovery using a proteomic approach 94
has several drawbacks. The techniques generally used for blood proteomics 95
(peptidomics) have technical limitation in terms of the analytical process, i.e., the 96
6
protocol requires removal of high-abundance plasma/serum proteins prior to analysis 97
for omitting undesirably disturbance for detection of small molecules like peptide. 98
This process likely resulted in some important DBMs being overlooked in some 99
cases.11,12 However, peptidomics analysis may be useful for DBM discovery, because 100
peptide fragments in blood show greater variation in total number and structure 101
compared with a genome/proteome.13 Therefore, sequestered peptides not detected (or 102
overlooked) to date by conventional peptidomic approaches may represent an important 103
source of candidate DBMs for subsequent clinical validation. Additionally, these 104
peptides will expect to provide novel pathophysiological information in terms of the 105
molecular composition of the circulation.11,14 106
We previously developed a one-step direct transfer technology for matrix assisted 107
laser desorption/ionization (MALDI) MS (BLOTCHIP®-MS)15 that does not require 108
reduction of protein concentrations in test samples prior to analysis. This technique 109
enabled detection of peptides in blood samples, including those that would otherwise be 110
adsorbed to blood proteins and so escape detection. Using this technology, we found 111
23 characteristic peptides as potential DBMs for PIH (including GH/PE as stated above) 112
in the serum of pregnant females; 7 of the 23 peptides were identified as fragments of 113
kininogen-1 (three peptides), α-2-HS-glycoprotein, fibrinogen-α, complement 114
component C4-A/B, and inter-α-trypsin inhibitor heavy chain H4 and thus to be 115
candidate DBMs for PIH.16 116
Because direct application of a method like peptidomic analysis is not suitable for 117
clinical screening, simple, reliable, and low-cost peptide quantitation in blood samples 118
is necessary for evaluation of the usefulness of peptides as DBMs. The aim of the 119
present study was to establish a simple and reliable quantitation system, sandwich 120
7
enzyme-linked immunosorbent assays (ELISA), for potential peptide DBMs for GH/PE 121
and to validate its clinical utility. Among the candidate peptides, three (two peptides 122
derived from kininogen-1, and one from α-2-HS-glycoprotein) were initially used as 123
target molecules based on their relatively high performance for PIH diagnosis, as 124
demonstrated in our preliminary study.16 125
126
8
Patients and Methods 127
128
Blood sample collection 129
130
The Ethic Committee board members of Juntendo University and Yamagata Saisei 131
Hospital approved the study protocol prior to the present study (registration #19-017 132
and #144, respectively). 133
Patients with hypertensive disorders were diagnosed according to the guidelines as 134
previously published.1,17 Sera were isolated essentially according to the method as 135
described previously,16 from pregnant females with hypertensive disorders (n = 34: GH; 136
n = 4 PE; n = 30)(Table 1), and healthy volunteers at 23/33 each gestational-week (n = 137
50: age (mean (SD); 32.8 (4.8) year-old at delivery). No protease inhibitor was used 138
for sample collection, since peptides in the body fluids are relatively stable during 139
sample preparation16. All blood samples were stored aliquots at -80°C until use and 140
not allowed to repeat freeze/thaw cycle more than twice. Written informed consents 141
were obtained from participants in each study. 142
143
Chemicals 144
145
BLOCK ACE® powder was purchased from DS Pharma Biomedical Co., Ltd (Osaka, 146
Japan). High-sensitivity streptoavidine-HRP was from Wako Pure Chemical Industries, 147
Ltd., Osaka, Japan. Bovine serum albumin (BSA)(fraction V) and SIGMAFASTTM 148
o-phenylenediamine dihydrochloride (OPD) tablets were purchased from 149
9
Sigma-Aldrich Co. LLC., St. Louis, MO, USA. All other chemicals were obtained 150
commercially and were of the highest purity available. 151
152
Polyclonal antibody (pAb) production for peptides 153
154
Potential biomarker candidate peptides for GH/PE (kininogen-1439-456, m/z 2081.00 155
termed as PDA039; kininogen-1438-456, m/z 2209.12 (PDA044); and cysteinyl 156
α2-HS-glycoprotein341-367, m/z 2858.61 (PDA071))16 were chemically synthesized. As 157
immunogens, the amino acid residues of each N-/C-terminal peptide (Supplementary 158
Table S1) conjugated with keyhole limpet haemocyanin (KLH) at their non-terminus 159
sites were used. 160
For pAbs production, rabbits were immunized subcutaneously with 0.15 mg of 161
KLH-conjugated peptides emulsified in complete Freund’s adjuvant. Two weeks after 162
the first injection, three subcutaneous booster injections of 0.3 mg antigen emulsified in 163
incomplete Freund’s adjuvant were performed every two-week. The rabbits were 164
sacrificed 20 days after the last immunization and the serum were isolated from total 165
bloods. 166
Anti-N-/C-terminal peptides pAbs were affinity purified from the isolated serum by 167
each immunogen-peptide coupled with cyanogen bromide-activated Sepharose 4B (GE 168
Healthcare Life Sciences, Uppsara, Sweden). A part of each pAb was biotinylated for 169
use as a secondary Ab in sandwich ELISA system for peptide quantitation (see below). 170
The production of pAbs for N-/C-terminal peptides described above was performed 171
commercially by Ab production custom services in Scrum Inc., Tokyo, Japan. 172
173
10
Sandwich ELISA for quantitation of the peptides 174
175
Each of the purified pAbs was coated on 96-well flat bottom 2HB plate (cat# 3455, 176
Thermo Fisher Scientific Inc., Waltham, MA, USA) in a volume of 100 µl of coating 177
buffer (pH 8.5) containing 0.017 M Na2B4O710H2O, 0.12 M NaCl for 2 hr at 37°C. 178
After blocking with 1% (w/v) BLOCK ACE® in ultra pure water for 1 hr at 37°C, 100 179
µl of sample solutions was added to each well and reacted for over 20 hr at 4°C. For 180
dilution of samples, 0.1% BSA in phosphate buffer saline (PBS) (pH 7.4) was used. 181
After washing with 0.4% (w/v) BLOCK ACE® solution containing 0.05% (v/v) 182
Tween-20 (washing solution) three times, the plate was treated with various 183
concentration of biotin-labeled secondary pAb (volume of each was 100 µl) in 0.4% 184
(w/v) BLOCK ACE® solution for over 20 hr at 4°C. At the end of reaction, each plate 185
was washed at least three times with washing solution, then each well was treated with 186
100 µl of 0.01% (v/v) high-sensitivity streptoavidine-horseradish peroxidase (HRP) in 187
PBS (pH 7.4) containing 0.25% (w/v) BSA and 0.05% (v/v) Tween-20 for 1 hr at 188
ambient temperature. The bound antibody was determined by reacting with OPD for 189
45 min at 37°C, then stopped by the addition of 100 μl 1 M H2SO4. The amount of 190
reaction product was read by absorbance at 492 nm using ELISA plate reader (Wallac 191
1420 ARVO MX, PerkinElmer Inc., Waltham, MA, USA) 192
193
Immunoaffinity-supported stable-isotope dilution MS quantitation 194
195
Preparation of anti-peptide pAb-conjugated beads Each pAb against 196
the N-terminal of PDA039, PDA044, and the C-terminal of the both peptides that 197
11
shared the same C-terminal sequence (Supplementary Table S1) was covalently 198
conjugated to N-hydroxysuccinimide-activated Sepharose 4B Fast Flow (GE 199
Healthcare) according to the manufacturer’s instruction. Each pAb (0.75 mg) was 200
mixed in 2.0 ml solution of 0.2 M NaHCO3 (pH 8.3), 0.5 M NaCl containing 1.0 ml of 201
the activated beads. The mixture was reacted for 2 hr at ambient temperature with 202
gentle swirling. After the incubation, complete immobilization of the pAbs was 203
confirmed by measuring protein concentration of the reaction mixture with Coomassie 204
Protein Assay reagent (Thermo Fisher). Three ml of 0.1 M 205
tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer (pH 8.5) was added to block 206
the residual active functional groups. The mixture was reacted for 30 min at ambient 207
temperature, and then continuously incubated overnight at 4°C. The pAb-immobilized 208
beads were washed for 5 times each with 0.1 M Tris-HCl (pH 8.5) and 0.1 M acetate 209
(pH 4.5) containing 0.5 M NaCl. Finally, the beads were equilibrated with 5 ml PBS 210
(pH 7.4) containing 0.1% (w/v) NaN3 and stored at 4°C until use. 211
212
Quantitation of potential DBM peptides (PDA039/044) in serum samples 213
with MS Stable isotope-labeled PDA039 (PDA039H21: 214
HNL(13C6,15N)GHG(13C2,
15N)HK(13C6,15N2)HERDQG(13C2,
15N)HGHQ) and PDA044 215
(PDA044H16: K(13C6,15N2)HNLGHGHK(13C6,
15N2)HERDQGHGHQ) were chemically 216
synthesized (Scrum). Absolute quantitation of PDA039/044 was conducted by 217
MALDI-TOF-MS in combination with stable-isotope dilution method using 218
PDA039H21/PDA044H16 and specific binding and elution of peptides with the 219
Ab-immobilized gel. Since the peptide isolation step was essential for precise 220
quantitation in this procedure, the gel immobilized with Ab against the C-terminal 221
12
sequence of PDA039/044 was utilized to capture non-labeled and stable-isotope labeled 222
peptides. Blood samples (totally 45 sera samples randomly selected from each group; 223
23/33-week healthy pregnant women and GH/PE patients; 15 samples each) were used 224
for the measurement. A serum sample (20 l; n = 2) was diluted with 960 l PBS (pH 225
7.4) in new 1.5 ml low-binding tube (Proteosave SS; Sumitomo Bakelite Co. Ltd., 226
Tokyo, Japan). Stable-isotope labeled peptide (10 l) (PDA039H21 or PDA044H16; 227
50 or 100 fmol/l, respectively) was added as an internal standard. After mixing with 228
inversion, the solution was immediately mixed with 5 l of pAb-conjugated Sepharose 229
4B Fast Flow gel (50% slurry). The mixtures were gently stirred for 1 hr and the gels 230
were spun down with centrifugation for 12,000 x g for 20 sec. The solution was 231
removed by vacuum aspiration. Subsequently, the gels were washed two times with 20 232
mM potassium phosphate, pH 7.2 containing 0.5 M KCl (for the purpose of 233
quantitation) or with PBS (pH 7.4) containing 0.5 M NaCl (for the others). Washing 234
the gels with potassium containing buffer converted part of the peptide to its 235
potassium-adduct (+43.964 u), which did not interfere with MS measurement of the 236
stable-isotope labeled peptide (PDA039H21/PDA044H16; +21.000/+16.000 u from the 237
unlabeled peptide, respectively)(Supplementary Figure S1). Finally the gels were 238
washed with ultra pure water. The antibody-bound peptides were eluted with 25 l of 239
0.1% trifluoroacetic acid (TFA) containing 10% acetonitrile for 5 min at ambient 240
temperature, and then centrifuged. The supernatant was collected as the eluate. 241
All mass spectra were obtained on an UltraFlex II TOF/TOF-MS (Bruker Daltonics, 242
Bremen, Germany) in the reflector mode (for quantitation) or in the linear mode (for the 243
others). Each analyte solution was mixed with saturated -cyano-4-hydroxycinnamic 244
acid in 0.1% TFA/acetonitrile (50:50) in the ratio of 1:2 and spotted (2 l per spot) in 245
13
duplicate onto MTP 384 Ground Steel TF target plate (Bruker Daltonics). 246
Standard samples were prepared for optimization of measurement conditions with 247
MS. According to the optimized conditions, the PDA039/044 concentration in the 248
subject sera was calculated with considering the isotope purity. For more detailed 249
descriptions of these matters, refer to Supplementary Methods, Figure S1, S2 and Table 250
S2. 251
252
Statistical Analysis 253
254
Statistical analyses were performed with R statistical environment software (R Core 255
Team, 2013; http://www.R-project.org/). Receiver operation characteristic (ROC) 256
analysis was conducted to determine the diagnostic performance of DBM candidate 257
peptide level in distinguishing GH/PE patients and healthy pregnant women with 258
package Epi (Carstensen et al, 2013; http://CRAN.R-project.org/package=Epi) within R 259
software. Area under the curve (AUC), sensitivity (SN), specificity (SP) and threshold 260
values (positive/negative predictive value; PPV/NPV) were calculated from ROC curve 261
as an indicator of the diagnostic value. The optimal cut-off thresholds for diagnosis 262
were determined according to Youden’s index.18 Confidence interval (CI) of 263
diagnostic values for AUC was calculated with the non-parametric bootstrapping 264
method of sample data. Mann-Whitney U-test for non-parametric data was used for 265
analysis between two groups of differences. A probability of P < 0.05 was considered 266
statistically significant. 267
268
14
Results 269
270
Conditions for peptide quantitation by sandwich ELISA 271
272
As an initial step, we identified the optimum sandwich ELISA conditions for 273
quantitation of the PDA039/044/071 synthetic peptides. Affinity-purified pAbs 274
against N-/C-terminal peptides at several concentrations were assessed as the captured 275
(primary) Ab. A biotinylated pAb against the opposite terminus of the peptide was 276
used as the detection (secondary) Ab. Two primary Ab concentrations, 5 and 10 µg/ml 277
diluted in PBS (pH 7.4) containing 0.1% (w/v) BSA were used, followed by 0.03 to 1 278
µg of each biotinylated secondary pAb. 279
Data obtained in the above preliminary experiments suggested that the optimum 280
conditions for PDA039/044 quantitation were as follows: primary Ab, anti-C-terminal 281
pAb for PDA039/044 at the concentration of 5 µg/ml; detection Ab, anti-N-terminal 282
biotinylated pAb for each peptide at 0.25 µg/ml. Using these conditions, typical 283
reaction curves for synthetic PDA039/PDA044 were obtained (Figure 1). The 284
standard curve was established within the range from 0.1 to 10 ng/ml for both PDA039 285
(Figure 1(a)) and PDA044 (Figure 1(b)) quantitation. Under these conditions, the 286
linear dynamic range was 0.5-5 ng/ml. Although similar standard curves were 287
obtained when synthetic peptides were spiked into standard serum diluted with PBS (pH 288
7.4) containing 0.01% BSA (Figure 1(c, d)), the optical densities at 492 nm were 289
somewhat shifted in the PDA039 quantitation (Figure 1(c)). 290
For PDA071 quantitation, however, the conditions above yielded no meaningful 291
analytical data. Because reasonable quantities of pAbs were obtained by affinity 292
15
purification using synthesized the N/C-terminal immunogen-peptide fragments 293
(Supplementary Table S1) immobilized on Sepharose 4B beads, the pAbs to PDA071 294
terminal peptides were likely unsuitable for the sandwich ELISA system. Using the 295
sandwich ELISA system with the conditions above, we examined the immunoreactivity 296
of the PDA071 pAbs using various detergents, including deoxycholic acid or low 297
concentration of sodium dodecyl sulfate;19 however, no significant pAb reactivity with 298
PDA071 was observed. Instead, the PDA071 anti-N-terminal pAb, but not the 299
anti-C-terminal, showed immnoreactivity only when the peptide was directly coated on 300
the ELISA plate (data not shown). 301
These data suggest that the steric structure of the C-terminus of PDA071 in nature is 302
somewhat different compared to that of the synthesized C-terminal peptide oligomer 303
(Supplementary Table S1) that was used to affinity purify of the pAb. Because the 304
anti-N-terminal pAb of PDA071 enabled quantitation of the peptide, we are currently 305
producing Abs against a more characteristic site of the candidate DBM, i.e., in the 306
vicinity of the cysteinyl site at α2-HS-glycoprotein358 (Supplementary Table S1). We 307
also aim to confirm the serum peptide concentration in PIH and healthy pregnant 308
females using stable isotope-labeled PDA071 by quantitative MS analyses. These 309
multidirectional approaches will enable clarification of the usefulness of PDA071 as a 310
DBM for GH/PE. 311
312
Specificity of pAbs against N-terminal PDA039/044 peptides 313
314
Among the biomarker candidate peptides for GH/PE that we identified previously, 315
both the PDA039 and 044 peptides originated from kininogen-1.16 In addition, 316
16
PDA044 possesses one additional amino acid residue at the N-terminus compared to 317
PDA039 (Supplementary Table S1). Therefore, we assessed the antigenic specificity 318
of both anti-N-terminal pAbs. Using conditions identical to those for quantitation of 319
the PDA039/044 sandwich ELISA systems (Figure 1), we evaluated the specificity of 320
each peptide detection system. As expected, the PDA039 detection system did not 321
cross-react with that of PDA044, and vice versa (Supplementary Figure S3). The MS 322
spectra of the eluates from anti-kininogen peptide antibody-immobilized beads indicate 323
that each anti-N-terminal Ab recognizes its appropriate antigen peptide, and the 324
anti-C-terminal Ab reacts specifically with the common terminal of PDA39/044 (Figure 325
2). 326
Taken together, these results indicate that the sandwich ELISA system is suitable for 327
detection of the PDA039/044 in serum. 328
329
Comparison between sandwich ELISA and quantitative MALDI-TOF-MS 330
analyses by Bland-Altman difference plots 331
332
The peptide (PDA039/044) concentrations in serum (randomly selected (n = 45) 333
from our serum stock) were assayed using the sandwich ELISA and MALDI-TOF-MS 334
systems. Each serum peptide level was plotted two-dimensionally; the correlation 335
assay values were as follows: PDA039; slope = 0.765, intercept = 1.665, and r2 = 0.898; 336
PDA044; slope = 0.570, intercept = 10.977, and r2 = 0.656 (Figure 3(a)). Although 337
PDA044 showed a relatively low correlation among these two assay systems, peptide 338
assay values converted by Bland-Altman difference plot20 with a correction method 339
reported by Dewett et al21 demonstrated that the most peptide concentrations provided 340
17
by ELISA and MS analysis were distributed within limits of agreement (LOA)(Figure 341
3(b)). 342
343
Measurement of serum PDA039/044 peptide levels by sandwich ELISA in 344
patients with GH/PE and normal pregnant females at 23/33 weeks of 345
gestation 346
347
Levels of the PDA039/044 peptides in serum from GH/PE patients and normal 348
pregnant females were examined using sandwich ELISA. The PDA039 level (median, 349
6.42; interquartile range (IQR), 3.35-16.62; n = 34) were significantly higher in the 350
serum of GH/PE patients compared to that of normal pregnant females at 23 (median, 351
2.18; IQR, 0.63-3.40; n = 50, P < 0.0001) and 33 (median, 1.08; IQR, 0.11-2.46; n = 50, 352
P < 0.0001) weeks of gestation (Figure 4(a)). It should be noted that a case of quite 353
high level of serum PDA39 was identified in the GH/PE patient group (Figure 4(a)). 354
This value was from PIH16 patient in Table 1. We confirmed the serum levels of 355
PDA039 peptides with GH/PE patients without the PIH16 case, but statistical 356
significance of PDA concentration was observed between GH/PE patients without 357
PIH16 and other groups (P < 0.0001; data not shown), like data showing in Figure 4. 358
Although the median PDA044 level in the serum from GH/PE patients (26.18 ng/ml) 359
was higher than that in the serum of normal pregnant females (medians, 15.25 and 20.64 360
ng/ml at 23 and 33 weeks of gestation, respectively), the PDA044 level in sera of all 361
subjects (GH/PE patients and normal pregnant females at 23 and 33 gestational weeks) 362
did not differ markedly among the three groups (Figure 4(b)). Seemingly, the serum 363
PDA044 concentration varied randomly during normal pregnancy from 23 to 33 364
18
gestational weeks (Figure 4(b)). 365
366
Diagnostic accuracy of PDA039 in patients with GH/PE 367
368
Since PDA039 has a potential utility as DBM for GH/PE (Figure 4), we evaluated its 369
diagnostic accuracy in the patients using ROC curve analysis. The ROC values of 370
serum PDA039 levels in GH/PE patients versus those in normal pregnant females were: 371
SN = 58.8%, SP = 96.0%, PPV = 90.9%, NPV = 77.4%, AUC (95%CI) = 0.844 372
(0.760-0.927), and cut-off value = 5.02 ng/ml (vs. 23 weeks of gestation; Figure 5(a)); 373
SN = 85.3%, SP = 86.0%, PPV = 80.6%, NPV = 89.6%, AUC (95%CI) = 0.890 374
(0.817-0.961), and cut-off value = 2.81 ng/ml (vs. 33 weeks of gestation; Figure 5(b)). 375
376
19
Discussion 377
378
Data obtained from present study suggested that PDA039, a peptide fragment 379
derived from kininogen-1, shows clinical usefulness as a DBM for at least diagnosis of 380
GH/PE (Figures 4(a) and 5), whereas PDA044 (also derived from kininogen-1) did not 381
(Figure 4(b)). In the inspection process of quantitation PDA044, we found that sodium 382
adduct (+22) of the peptide (PDA039/PDA044) was overlapped with the stable-isotope 383
labeled peptide (PDA039H21/PDA044) in the linear mode of MALDI-TOF-MS. For 384
the purpose of removing the sodium-adduct interference, we selected potassium 385
containing buffer as a washing buffer instead of PBS with sodium chloride and 386
measured peptides in the reflector mode for peptide quantitation. In addition, 387
stable-isotope labeling efficiency of the synthesized peptide 388
(PDA039H21/PDA044H16) affected the quantitative value of the peptides. Therefore, 389
we calculated accurate height values resulted in more accurate measurement of the 390
peptides (see Supplementary Methods). However, the absolute values of quantitation 391
for PDA044 were somewhat different between ELISA and MS methods. According to 392
our preliminary experimental data, PDA044 as well as PDA039 possess biding affinity 393
to serum major proteins such as albumin and IgG. PDA044 has much high affinity, 394
approximately 1.5-fold affinity to serum protein compared to that of PDA03916. 395
Although PDA044 possesses only one additional amino acid residue at the N-terminus 396
compared to PDA039 (Supplementary Table S1), the detection ELISA system for 397
PDA044 did not show cross-reactivity for that of PDA039 (Supplementary Figure S3). 398
This implies that the pAb against N-terminus of PDA044 has quite high specificity to 399
the epitope. Based on the experimental data, we speculate that the reason why the 400
20
absolute values between quantitative MS analysis and that by ELISA in the case of 401
PDA044 might be due to the effect of partial inhibition by serum unknown factor(s) 402
including abundant proteins on the immunoreaction of the pAb to N-terminus of 403
PDA044 in the ELISA system. 404
As a functional peptide derived from kininogen-1, bradykinin regulates blood 405
pressure via the nitric oxide signaling pathway in vascular smooth muscle.22 406
Conversely, angiotensin-converting enzyme, a key molecule in the pathway of 407
renin-angiotensin-aldosterone pathway, has an alternative activity as kininase II (a 408
peptidase) that inactivates bradykinin.23 These results imply that hyper- and 409
hypo-tensive mechanisms display both complex and multidirectional molecular 410
interaction. Indeed, an imbalance between the levels of vasoconstrictor and 411
vasodilator substances in the placenta, including kininogen, in patients with PIH was 412
reported.24 However, the pathophysiology of patients with hypertensive disorders of 413
pregnancy remains unclear, and the physiological functions of circulating peptides 414
(including fragments derived from kininogen-1), other than bradykinin for blood 415
pressure, are far from being completely understood. 416
Our previous study revealed that molecular mechanism as to production of 417
PDA039/044 might be different from that of bradykinin from kininogen-1, since no 418
significant difference in serum bradykinin level was observed in between the patients 419
and control groups.16 Therefore, monitoring of these peptide-fragments derived from 420
kininogen-1 using the simple quantitation method developed in the present study may 421
provide a new insight into pathophysiology of GH/PE. Recent studies of the serum 422
peptidome have suggested that clinical serum peptidomic analyses reflect various 423
pathological disorders involving systematic changes, including hypertensive disorders 424
21
of pregnancy (for review,25,26). Numerous peptides in serum are considered to be not 425
simply products of protein degradation. Several peptides have been identified to have 426
biological activity as hormones, growth factors, neurotransmitters, and antibiotic 427
agents.27 As a biological event, it is generally considered that the contentious stressful 428
environment induces variety of stressors, and they sometimes cause pathophysiological 429
changes of organs resulting in various disorders. Because production of peptide 430
fragments in the blood is thought to regulate cellular metabolism, the homeostatic 431
balance in between proteases and their inhibitors may be essential to maintain a healthy 432
conditions; this is also likely to be the case in pregnancy. 433
434
22
Conclusion 435
436
The present study clearly shows the clinical usefulness of PDA039 as a DBM for 437
GH/PE using a simple quantitation system. The DBM candidates we identified in the 438
previous study are quite unique molecules by specific plate, BLOTCHIP® for the 439
MALDI-TOF/MS system16. Using this novel technology, the DBM candidates 440
identified in our previous study have positive potential as the DBM biomarkers 441
compared to that identified by conventional omics methods26. Therefore, the special 442
conclusion of the present study is not only the establishment of the simple quantitation 443
technology by the ELISA, but also clinical utility of PDA039, at least, is validated using 444
clinical samples. Although development of a simple quantitation system for other 445
potential DBM peptides16 is necessary, monitoring of the serum PDA039 concentration 446
during pregnancy would provide novel information regarding the pathophysiology of 447
GH/PE. Further multidimensional evaluation in combination with PDA039 and other 448
peptides, or other proposed DBMs for PE, such as placental growth factor, soluble 449
fms-like tyrosine kinase 1 receptor, and soluble endoglin28-30 would enhance our 450
understanding of the molecular mechanisms underlying, and provide information 451
regarding the clinical manifestations for hypertensive disorders of pregnancy. 452
453
23
REFERENCES 454
455
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Pressure in Pregnancy. Report of the National High Blood Pressure Education 457
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2. Mahmoudi N, Graves SW, Solomon CG, Repke JT, Seely EW. Eclampsia: 13-year 460
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United Kingdom. Am J Obstet Gynecol 2000; 182: 760-766. 464
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6. Savino R, Paduano S, Preianò M, Terracciano R. The proteomics big challenge for 472
biomarkers and new drug-targets discovery. Int J Mol Sci 2012; 13: 473
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7. Richter R, Schulz-Knappe P, Schrader M, et al., Composition of the peptide 475
fraction in human blood plasma: database of circulating human peptides. J 476
Chromatogr B Biomed Sci Appl 1999; 726: 25–35. 477
24
8. Schrader M, Schulz-Knappe P. Peptidomics technologies for human body fluids. 478
Trends Biotechnol 2001; 19 (10 Suppl): S55-60. 479
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Characterization of the low molecular weight human serum proteome. Mol Cell 481
Proteomics 2003; 2: 1096-1103. 482
10. Schulz-Knappe P, Schrader M, Zucht HD. The peptidomics concept. Comb 483
Chem High Throughput Screen 2005; 8: 697-704. 484
11. Lowenthal MS, Mehta AI, Frogale K, et al. Analysis of albumin-associated 485
peptides and proteins from ovarian cancer patients. Clin Chem 2005; 51: 486
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13. Schulz-Knappe P, Zucht HD, Heine G, Jürgens M, Hess R, Schrader M. 491
Peptidomics: the comprehensive analysis of peptides in complex biological 492
mixtures. Comb Chem High Throughput Screen 2001; 4: 207-217. 493
14. Hashiguchi T, Tanaka K, Lee L-J, et al. Diagnostic value of serum peptidome 494
analyses for protease activated pathological conditions beyond cancer diagnosis. 495
Med Hypotheses 2009; 73: 760-763. 496
15. Tanaka K, Tsugawa N, Kim YO, Sanuki N, Takeda Y, Lee L-J. A new rapid and 497
comprehensive peptidome analysis by one-step direct transfer technology for 1-D 498
elecrophoresis/MALDI mass spectrometry. Biochem Biophys Res Commun. 2009; 499
379: 110-114. 500
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16. Araki Y, Nonaka D, Tajima A, et al. Quantitative peptidomic analysis by a newly 501
developed one-step direct transfer technology without depletion of major blood 502
proteins: Its potential utility for monitoring of pathophysiological status in 503
pregnancy-induced hypertension. Proteomics 2011; 11: 2727-2737. 504
17. American College of Obstetricians and Gynecologists; Task Force on Hypertension 505
in Pregnancy. Hypertension in pregnancy. Report of the American College of 506
Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy. 507
Obstet Gynecol, 2013; 122: 1122-1131. 508
18. Youden WJ. Index for rating diagnostic tests. Cancer 1950; 3: 32-35. 509
19. Lechtzier V, Hutoran M, Levy T, Kotler M, Brenner T, Steinitz M. Sodium 510
dodecyl sulphate-treated proteins as ligands in ELISA. J Immunol Methods 2002; 511
270: 19-26. 512
20. Bland JM, Altman DG. Statistical methods for assessing agreement between two 513
methods of clinical measurement. Lancet 1986; 1(8476): 307-310. 514
21. Dewitte K, Fierens C, Stöckl D, Thienpont LM. Application of the Bland-Altman 515
plot for interpretation of method-comparison studies: a critical investigation of its 516
practice. Clin Chem 2002; 48: 799-801. 517
22. Berguer R, Hottenstein OD, Palen TE, Stewart JM, Jacobson ED. 518
Bradykinin-induced mesenteric vasodilation is mediated by B2-subtype receptors 519
and nitric oxide. Am J Physiol 1993; 264: G492-496. 520
23. Erdös EG. Conversion of angiotensin I to angiotensin II. Am J Med 1976; 60: 521
749-759. 522
24. Mohamed M, Larmie ET, Singh HJ, Othman MS. Tissue kallikrein and kininogen 523
levels in fetoplacental tissues from normotensive pregnant women and women 524
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with pregnancy-induced hypertension. Eur J Obstet Gynecol Reprod Biol 2007; 525
137: 15-19. 526
25. Tammen H, Peck A, Budde P, Zucht HD. Peptidomics analysis of human blood 527
specimens for biomarker discovery. Expert Rev Mol Diagn 2007; 7: 605-613. 528
26. Araki Y, Nonaka D, Hamamura K, et al. Clinical peptidomic analysis by a 529
one-step direct transfer technology: Its potential utility for monitoring of 530
pathophysiological status in female reproductive system disorders. J Obstet 531
Gynecol Res 2013; 39: 1440-1448. 532
27. Tinoco AD, Saghatelian A. Investigating endogenous peptides and peptidases 533
using peptidomics. Biochemistry 2011; 50: 7447-7461. 534
28. Maynard SE, Min J-Y, Merchan J, et al. Excess placental soluble fms-like 535
tyrosine kinase 1 (sFlt-1) may contribute to endothelial dysfunction, hypertension, 536
and proteinuria in preeclampsia. J Clin Invest 2003; 111: 649-658. 537
29. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic factors and risk of 538
preeclampsia, New Engl J Med 2004; 350: 672-683. 539
30. De Vivo A, Baviera G, Giordano D, Todarello G, Corrado F, D'anna R. 540
Endoglin, PIGF and sFlt-1 as markers for predicting pre-eclampsia. Acta Obstet. 541
Gynecol Scand 2008; 87: 837-842. 542
543
27
Figure legends 544
545
Figure 1. Peptide quantitation of PDA039/044 peptides by sandwich ELISA system. 546
Typical metage lines for standard synthetic peptides. Anti-C-terminal affinity purified 547
PDA039/044 pAbs (5 µg/ml) were coated as primary Abs, respectively. Each 548
anti-N-teminal peptide biotinyl pAb (PDA039 (a); PDA044 (b)) was used as a detection 549
Ab. Chemically synthetic peptides were diluted in PBS (pH 7.4) containing 0.1% BSA 550
and their absorbance values were monitored. Open circles, PDA039; Closed circle, 551
PDA044. Identical sandwich ELISA system for the detection of peptides in 20-fold (c) 552
or 200-fold (d) diluted serum with PBS (pH 7.4) containing 0.01% BSA. ELISA: 553
enzyme-linked immunosorbent assay; pAb: polyclonal antibody; BSA: bovine serum 554
albumin; PBS: phosphate buffer saline 555
556
Figure 2. MS analysis of eluates from anti-kininogen peptide antibody immobilized 557
beads. The beads were mixed with sera from GH/PE patients, washed, and bound 558
peptides were eluted. Two pairs of anti N-terminal and C-terminal kininogen peptide 559
antibodies were found to bind respective antigen peptides. Bound kininogen peptides 560
were eluted from immobilized anti-PDA039 N-terminal pAb (a), anti-PDA039/044 561
C-terminal pAb (b), anti-PDA044 N-terminal Ab (c) on Sepharose 4B Fast Flow beads, 562
and beads only (negative control)(d). Closed circles indicate peaks that also found 563
when control beads (no-Ab immobilized beads) were used instead. Open circles 564
indicate mono-(+22u), di-(+44u) or tri-(+66u) sodiated peptides. GH/PE: gestational 565
hypertension/preeclampsia; pAb: polyclonal antibody 566
28
567
Figure 3. Comparison of serum peptide concentrations assayed by the sandwich 568
ELISA and MALDI-TOF-MS systems. Two-dimensional correlation of assay values 569
between the peptide concentration by ELISA and that by MALDI-TOF-MS (a), and 570
Bland-Altman plots comparing data obtained from both assay systems (b). PDA039: 571
mean difference (solid line) = 0.583, +LOA/-LOA (dotted lines) = 2.214/-1.047, and 572
PDA044: mean difference = 0.065, +LOA/-LOA = 1.711/-1.581, respectively. ELISA: 573
enzyme-linked immunosorbent assay; MALDI-TOF-MS: matrix assisted laser 574
desorption/ionization time-of-flight mass spectrometry; LOA: limits of agreement 575
576
Figure 4. Serum concentration of disease biomarker candidate peptides for GH/PE by 577
sandwich ELISA. Concentration of PDA039 (a) and 044 (b) in the pregnant women as 578
shown by box plots. Open/closed circles indicate peptide concentration in the healthy 579
pregnant women and patients with GH/PE, respectively. Change of the peptide 580
concentrations within a pair serum is represented by dotted lines. Asterisks indicate 581
significant differences statistically between two groups. GH/PE: gestational 582
hypertension/preeclampsia; ELISA: enzyme-linked immunosorbent assay 583
584
Figure 5. Diagnostic accuracy of PDA039 for GH/PE. ROC curves of PDA039 in 585
patient with GH/PE versus normal pregnant women at 23-(a) or 33-week of gestation 586
(b). SN, SP, PPV, and NPV were calculated as described in the “Materials and 587
Methods”. ROC: receiver operation characteristic; GH/PE: gestational 588
hypertension/preeclampsia; SN/SP: sensitivity/specificity; PPV/NPV: positive/negative 589
predictive value 590
Figure 1 Hamamura et al.
⊿A
bso
rban
ce a
t 49
2 n
m
Peptide concentration (ng/ml)
(a)
(c)
(b)
(d)
0 0.1 1.0 10 100
0 0.1 1.0 10 100
0.5
0.4
0.3
0.2
0.1
0
0.5
0.4
0.3
0.2
0.1
00 0.1 1.0 10 100
0 0.1 1.0 10 100
1.0
0.8
0.6
0.4
0.2
0
1.0
0.8
0.6
0.4
0.2
0
Inte
nsi
ty (
a.u
.)(x
105 )
m/z
(a)
(b)
(c)
2050 2100 2150 2200 2250
0.6
0.4
0.2
0
0.8
0.4
01.0
0.5
0
PDA039 PDA044
Figure 2 Hamamura et al.
0 20 40 60 80 100 120
100
80
60
40
20
0
EL
ISA
ass
ay (
ng
/ml)
MA
LD
I-M
S –
EL
ISA
ass
ay
(% o
f av
erag
e)
4
3
2
1
0
-1
-2
-30 20 40 60 80 100 120
140
120
100
80
60
40
20
00 20 40 60 80 100 120 140
MALDI-MS (ng/ml)
4
3
2
1
0
-1
-2
-3
Average of two methods (ng/ml)
r2 = 0.656
r2= 0.898
PDA039 PDA039
PDA044PDA044
2.214
-1.047
0.583
1.711
-1.581
0.065
Figure 3 Hamamura et al.
0 20 40 60 80 100 120
(a) (b)
200
150
100
50
0
80
60
40
20
0
Figure 4 Hamamura et al.
(a)S
eru
m c
on
cen
trat
ion
(n
g/m
l)
23-week 33-weekNormal pregnancy
GH/PE
(b)
Ser
um
co
nce
ntr
atio
n (
ng
/ml)
23-week 33-weekNormal pregnancy
GH/PE
P > 0.0001
P > 0.0001
Figure 5 Hamamura et al.
1.0
0.8
0.6
0.4
0.2
0.0
(a)
False positive rate0.0 0.2 0.4 0.6 0.8 1.0
Tru
e p
osi
tive
rat
e
SN: 58.8%SP: 96.0%PPV: 90.9%NPV: 77.4%
Cutoff value: 5.02 ng/mlAUC : 0.844
1.0
0.8
0.6
0.4
0.2
0.0
(b)
False positive rate0.0 0.2 0.4 0.6 0.8 1.0
Tru
e p
osi
tive
rat
e
SN: 85.3%SP: 86.0%PPV: 80.6%NPV: 89.6%
Cutoff value: 2.81 ng/mlAUC : 0.890
Table 1. Clinical profiles of sera isolated from pregnant patients with hypertensive disorders ID *Age Gestational #BMI §Diagnosis §Blood pressure ‡Medication **Other week level duration (days) PIH 1 33 32 32.8 PE severe - - PIH 2 36 30 20.0 PE mild methyldopa (12) - PIH 3 32 31 20.1 PE mild methyldopa (4) - PIH 4 37 36 22.2 PE mild - - PIH 5 31 31 u/k PE severe - - PIH 6 33 35 33.3 PE severe - - PIH 7 39 37 17.9 PE severe nicardipine (3) - PIH 8 31 39 u/k PE severe - - PIH 9 32 35 u/k PE (HELLP) severe - hypertension PIH 10 31 31 22.3 PE severe nicardipine (2) - PIH 11 25 34 21.8 PE mild - - PIH 12 35 37 36.0 PE mild methyldopa (6) - PIH 13 34 30 23.4 PE severe - - PIH 14 23 38 32.6 GH mild - - PIH 15 40 38 17.7 PE severe - - PIH 16 35 38 22.1 PE mild hydralazine -
hydrochloride (u/k)
PIH 17 38 30 24.4 PE mild - - PIH 18 39 34 20.0 PE mild - - PIH 19 40 36 31.2 PE severe - - PIH 20 33 29 22.6 PE severe - - PIH 21 41 34 25.6 GH mild methyldopa - /nicardipine (29) PIH 22 29 36 27.2 PE mild - - PIH 23 28 38 24.2 PE severe methyldopa (11) - PIH 24 30 39 21.4 GH severe - Type1 DM PIH 25 39 27 u/k PE (superimposed) severe methyldopa (122) hypertension PIH 26 21 38 19.9 PE mild - - PIH 27 41 38 26.6 PE severe hydralazine - hydrochloride (u/k) PIH 28 24 36 21.6 PE severe hydralazine - hydrochloride (2) PIH 29 34 34 24.1 PE severe - - PIH 30 38 35 27.9 GH severe - - PIH 31 38 31 21.2 PE severe nicardipine (9) - PIH 32 34 34 22.3 PE (HELLP) severe methyldopa hypertension /MgSO2 (4) PIH 33 43 29 27.1 PE (superimposed) severe methyldopa (45) hypertension PIH 34 30 32 32.8 PE (superimposed) severe methyldopa (9) hypertension Mean 33.7 34.2 24.6 19.8 (SD) (5.5) (3.4) (5.0) (33.1) u/k, unknown; -, negative; PIH, pregnancy induced hypertension; PE, preeclampsia; GH, gestational hypertension; DM, diabetes mellitus; SD, standard deviation *Age of patient at delivery #Body mass index (BMI) before pregnancy §According to the clinical criteria for hypertensive disorders of pregnancy.1,5,17 ‡Indicates days of anti-hypertensive medication before sample collection **Medical history of patient before pregnancy
1
Supplementary Methods
Optimization of peptide measurement for human sera
Human sera (Millipore Corporation, Billerica, MA, USA)(20 l) were spiked with
known amount of peptides: a fixed concentration of PDA039H21/PDA044H16 (0.5
pmol and 1.0 pmol, respectively) and various amounts of PDA039/044 (0, 0.10, 0.21,
0.42, 0.83, and 1.67 pmol for PDA039, and 0, 0.31, 0.63, 1.25, 2.50, and 5.00 pmol for
PDA044). The sample was prepared in duplicate. The spiked serum was treated with
the pAb-conjugated Sepharose 4B Fast Flow gel. Subsequently, the bound peptides
were eluted and spotted onto a MALDI-target plate as described above. The optimal
laser power was set according to the following criteria: a) the peaks of the standard
peptides are apparently not saturated even at the highest amount but give the maximal
intensities, and b) peptide intensity ratios and their concentrations are in the linear
correlation (Supplmentary Figure S2). Each mass spectrum for peptide quantitation
was obtained by accumulation of 40 single spectra (50 laser shots/spectrum × 2
spectra/spot × 20 varying position within a spot). All spectra were processed
(smoothing and baseline-subtraction) and analyzed with FlexAnalysis 2.4 software
(Bruker Daltonics). The monoisotopic peak of the peptide (PDA039, PDA044,
PDA039H21, or PDA044H16) was automatically picked and the height value of the
peptide was extracted using the APEX algorithm within the software. For quantitation
of the peptide, four height values obtained from two spots per sample, which was
doubly prepared, was used for calculation.
Calculation for the ratio of the non/stable isotope labeled peptide
Concentration of PDA039/PDA044 in the subject sera was calculated from the
monoisotopic peak height ratio of PDA039/PDA044 to that of
2
PDA039H21/PDA044H16 as follows: An isotopic peak distribution of the non-labeled
(light) peptide significantly differs from the stable-isotope labeled (heavy) peptide
(Supplementary Table S2); given equal amount of the light and heavy peptides, fraction
of monoisotopic peak of PDA039 ([M+H]+ = 2080.967) is 0.322 and that of
PDA039H21 ([M+H]+ = 2101.952) is 0.390 (Supplementary Table S2). Therefore, the
ratio of PDA039 to PDA039H21 is calculated to be 0.322/0.390 = 0.826. However,
the isotopic labeling efficiency of SI-labeled amino acid for heavy peptides synthesis
was 98% in this study. Since “heavy” PDA039H21 has four SI-labeled amino acids as
building blocks (one leucine and lysine, and two glycines), PDA039H21 is not pure in
terms of SI-labeling. In this sense, the purity of PDA039H21 is calculated to be
(0.98)4 = 92.2%. As judged by the MALDI-MS result (Supplementary Figure S1), a
major impurity of PDA039H21 was a peptide with a monoisotopic peak of [M+H]+ =
2100.95, which was 1 u smaller than completely labeled PDA039H21 ([M+H]+ =
2101.95). For the purpose of removing the influence of this impurity on quantitation
of the peptide, the height of the second isotopic peak ([M+H]+ = 2101.95) of the
impurity was subtracted from that of the monoisotopic peak of PDA039H21 ([M+H]+ =
2101.95). Since the isotopic distribution of this impurity (only 1 u smaller than
PDA039H21) was virtually equal to the completely labeled PDA039H21, a fraction of
monoisotopic peak of PDA039H21 was recalculated to be {0.390 × 0.922 (purity of
PDA039H21)} – {(1-0.922) × 0.348 (fraction of the second isotopic peak of the
impurity)} = 0.332. When the recalculated fraction of the monoisotopic peak of
PDA039H21 was used, the ratio of PDA039 to PDA039H21 described above was also
recalculated to be 0.322/0.332 = 0.97. In a similar manner, fraction of the
monoisotopic peak of PDA044H16 was calculated to be 0.317, so that the ratio of
PDA044 to PDA044H16 is 0.299/0.317 = 0.941. We used these fraction values of the
monoisotopic peak of the heavy peptides for calculation of the ratio in this study.
Supplementary Figure legends
Supplementary Figure S1. Quantitation of the peptide PDA039/PDA044 using
pAb-conjugated gel assisted MS. Different combinations of the peptides were spiked
into control sera and the peptides were measured with the pAb-conjugated gel assisted
MALDI-TOF-MS analysis. A control serum was spiked with (a) 50 fmol/l PDA039
and 100 fmol/l PDA044, (b) 50 fmol/l PDA039H21 and 100 fmol/l PDA044H16, or
(c) 50 fmol/l each of PDA039 and PDA039H21, and 100 fmol/l each of PDA039 and
PDA039H21. Concentration of the PDA039/PDA044 was calculated according to the
method described in the "Patients and methods" section. Any peak interfered to the
3
ratio calculation was not observed when either PDA039/PDA044 (a) or
PDA039H21/PDA044H16 (b) was spiked alone. In addition, potassium-adduct peak
(+22 u) also did not interfere with measurement of PDA039H21/PDA044H16. pAb:
polyclonal antibody; MALDI-TOF-MS: matrix assisted laser desorption/ionization
time-of-flight mass spectrometry
Supplementary Figure S2. The relationship between peptide-concentration and the
ratio of unlabeld/labeld peptides. In the case of PDA039 (a) and PDA044 (b).
Before quantitation of unknown samples, the laser power of the MALDI-TOF-MS
instrument was adjusted so that a linearity between the ratio of PDA039/PDA039H21,
(or PDA044/PDA044H16) was obtained. MALDI-TOF-MS: matrix assisted laser
desorption/ionization time-of-flight mass spectrometry
Supplementary Figure S3. Specificity of the peptide quantitation by sandwich
ELISA system. Quantitation systems of PDA039 (a) and PDA044 (b). Ab
concentrations used in the ELISA system were identical as shown in Figure 1.
Chemically synthetic peptides, PDA039 (open circles) and PDA044 (closed circles)
were diluted PBS (pH 7.4) containing 0.1% BSA. Typical metage lines for standard
synthetic peptides are presented. ELISA: enzyme-linked immunosorbent assay; BSA:
bovine serum albumin; PBS: phosphate buffer saline.
1
Supplementary Figure legends
Supplementary Figure S1. Quantitation of the peptide PDA039/PDA044 using
pAb-conjugated gel assisted MS in the reflector mode. Different combinations of the
peptides were spiked into control sera and the peptides were measured with the
pAb-conjugated gel assisted MALDI-TOF-MS analysis. A control serum was spiked
with (a) 50 fmol/l PDA039 and 100 fmol/l PDA044, (b) 50 fmol/l PDA039H21 and
100 fmol/l PDA044H16, or (c) 50 fmol/l each of PDA039 and PDA039H21, and 100
fmol/l each of PDA039 and PDA039H21. Concentration of the PDA039/PDA044
was calculated according to the method described in the "Patients and methods" and
"Supplementary Methods" sections. Any peak interfered to the ratio calculation was
not observed when either PDA039/PDA044 (a) or PDA039H21/PDA044H16 (b) was
spiked alone. In addition, potassium-adduct peak (+22 u) also did not interfere with
measurement of PDA039H21/PDA044H16. pAb: polyclonal antibody;
MALDI-TOF-MS: matrix assisted laser desorption/ionization time-of-flight mass
spectrometry
Supplementary Figure S2. The relationship between peptide-concentration and
the ratio of unlabeld/labeld peptides. In the case of PDA039 (a) and PDA044 (b).
Before quantitation of unknown samples, the laser power of the MALDI-TOF-MS
instrument was adjusted so that a linearity between the ratio of PDA039/PDA039H21,
(or PDA044/PDA044H16) was obtained. MALDI-TOF-MS: matrix assisted laser
desorption/ionization time-of-flight mass spectrometry
Supplementary Figure S3. Specificity of the peptide quantitation by sandwich
ELISA system. Quantitation systems of PDA039 (a) and PDA044 (b). Ab
concentrations used in the ELISA system were identical as shown in Figure 1.
Chemically synthetic peptides, PDA039 (open circles) and PDA044 (closed circles)
were diluted PBS (pH 7.4) containing 0.1% BSA. Typical metage lines for standard
synthetic peptides are presented. ELISA: enzyme-linked immunosorbent assay; BSA:
bovine serum albumin; PBS: phosphate buffer saline.
Inte
nsi
ty(x
104
)
2060 2080 2100 2120 2140 2160 2180 2200 2220 2240
m/z
PDA039
PDA039H21 (+21)
PDA044
PDA044H16 (+16)
(a)
(b)
(c)
K+ adducts
Supplementary Figure S1 Hamamura et al.
1.0
2.0
3.0
4.0
5.0
0
1.0
2.0
3.0
4.0
0
1.0
2.0
3.0
4.0
0
0 1 2 3 4
2.0
1.5
1.0
0.5
0
6.0
5.0
4.0
3.0
2.0
1.0
00 1 2 3 4 5 6
(a) (b)
Pep
tid
e-co
nce
ntr
atio
n (
pm
ol)
PDA039/PDA039H21 PDA044/PDA044H16
y = 0.4937x + 0.02
R2 = 0.9996
y= 1.0016x + 0.0513
R2 = 0.9996
Supplementary Figure S2 Hamamura et al.
Pep
tid
e-co
nce
ntr
atio
n (
pm
ol)
Supplementary Figure S3 Hamamura et al.
⊿A
bso
rban
ce a
t 49
2nm
Peptide concentration (ng/ml)
(b)1.2
0.8
0.4
0.00.001 0.1 10 1000
0.6
0.4
0.2
0.0
(a)
0.001 0.1 10 1000
Supplementary Table S1: Sequence of potential marker peptides for gestational hypertension/preeclampsia
_____________________________________________________________________________________________________________
Peptide ID Observed Origin of Amino acid Amino acid sequence
monoisotopic the peptide number
[M+H]+
_____________________________________________________________________________________________________________
PDA039 2081.00 Kininogen-1 439-456 a)HNLGHGHKHERDQGHGHQb)
PDA044 2209.12 Kininogen-1 439-456 a)KHNLGHGHKHERDQGHGHQb)
PDA071 2858.61 (Cysteinyl) α2- 341-367 a)TVVQPSVGAAAGPVVPPC(+Cys)PGRIRHFKVCb)
HS-glycoprotein
_____________________________________________________________________________________________________________
Amino acid sequences as an immunogen for the production of polyclonal antibody against each peptide portion are underlined
(N-terminus: a)) or shadowed (C-terminus; b)), respectively.
1
Supplementary Table S2: Isotopic peak distribution of peptides.
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PDA039
[M+H]+ Fraction
2080.967 0.322
2081.967 0.349
2082.967 0.205
2083.967 0.085
2084.967 0.028
2085.967 0.008
2086.967 0.002
2087.967 0
2086.967 0
PDA039H21
[M+H]+ Fraction
2101.952 0.390
2102.952 0.348
2103.952 0.175
2104.952 0.063
2105.952 0.018
2106.952 0.005
2107.952 0.001
2108.952 0
PDA044
[M+H]+ Fraction
2209.062 0.299
2210.062 0.346
2211.062 0.215
2212.062 0.095
2213.062 0.033
2214.062 0.009
2215.062 0.002
2216.062 0.001
2217.062 0
PDA044H16
[M+H]+ Fraction
2225.050 0.345
2226.050 0.350
2227.050 0.195
2228.050 0.078
2229.050 0.025
2230.050 0.007
2231.050 0.002
2232.050 0
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The values were calculated using Molecular Weight Calculator for Windows Ver 6.49
(http://www.alchemistmatt.com/mwtwin.html#availability).
