Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation

Figures & data

Figure 1.  Diagram of the iTRAQ workflow. The sequence of the workflow is indicated by the arrows in the right side. Pooled reference: Small aliquots from each of the 75 samples of amniotic fluid were pooled to create a reference sample. Deplete-concentrate-reduce-block: The amniotic fluid samples were depleted of albumin and IgG, subsequently concentrated and reduced. Digestion: The samples were subjected to trypsin digestion. Labeling: The reference sample and three individual samples (denoted A, B, and C in the figure) were labeled with iTRAQ reagents (the numbers 114–117 refer to the reporter mass of the individual iTRAQ reagent). Mixing and fractionation: The four labeled samples were mixed and subjected to strong cation exchange (SCX) fractionation to simplify the mixture prior to conducting liquid chromatography and mass spectrometry (LC-MS/MS). Each SCX fraction was subjected to LC-MS/MS. Liquid chromatography: Proteins were separated by liquid chromatography. The retention time is displayed in the horizontal axis. MS1: The first stage of mass spectrometry for a specific retention time is indicated schematically. Each peak in the mass spectrum (horizontal axis is m/z) corresponds to an individual peptide. MS2: A single peak is selected for collisional dissociation and the fragments subjected to a second stage mass spectrometry (tandem MS/MS). The reporter ions from the iTRAQ label appear in the low mass range, distinct from peptide fragment peaks. The pattern of peaks in the final mass spectrum is matched to a database in order to identify the parent peptide. The intensities of the reporter ion peaks indicate the relative abundance of the four samples.

Table I.  Demographic and clinical characteristics of patients with spontaneous preterm labor with intact membranes.

	PTL without IAI, term delivery (n = 26)	p	PTL without IAI, preterm delivery (n = 25)	p^a	PTL with IAI, preterm delivery (n = 24)	p^b
Maternal age (years)	21 (16–38)	NS	28 (16–45)	NS	23 (17–41)	NS
Parity	1 (0–5)	NS	1 (0–4)	NS	1 (0–4)	NS
Previous preterm delivery	15.4 (4/26)	NS	36 (9/25)	NS	13 (3/23)	NS
GA at amniocentesis (weeks)	29.4 (24–33.7)	NS	30 (23.4–33.4)	NS	30 (23.1–33.3)	NS
AF WBC count (cells/mm^3)	2 (0–35)	NS	3 (0–18)	<0.001	325 (0–9000)	<0.001
AF IL-6 (ng/ml)	0.6 (0.05–1.3)	0.009	1.1 (0.1–2.5)	<0.001	57.9 (5.6–246.8)	<0.001
Tocolysis	34.6 (9/26)	NS	48 (12/25)	NS	29.2 (7/24)	NS
Amniocentesis-to-delivery interval (days)	65 (33–109)	<0.001	6 (0–7)	0.02	1 (0–7)	<0.001
GA at delivery (weeks)	38.6 (37.3–40.1)	<0.001	31 (23.4–33.9)	NS	30.1 (23.1–34)	<0.001
Birth weight (g)	3045 (2580–4230)	<0.001	1630 (550–1990)	NS	1380 (660–2490)	<0.001

Values expressed as percentage (number) or median (range).

p, comparison between PTL who delivered at term and PTL who deliver preterm without IAI.

p^a, comparison between PTL who delivered preterm without IAI and PTL with IAI.

p^b, comparison between PTL who delivered at term and PTL with IAI.

PTL, preterm labor; IAI, intra-amniotic infection/inflammation; GA, gestational age; AF, amniotic fluid; WBC, white blood cell; IL, interleukin; NS, not significant.

Table II.  Proteins previously reported in the literature to be differentially up-regulated in the amniotic fluid of patients with IAI.

Protein name in present study	Protein name as cited	Reference
CAPG macrophage-capping protein	Macrophage capping protein	[18]
LCN2 neutrophil gelatinase-associated lipocalin precursor	Neutrophil gelatinase-associated lipocalin	[18]
MPO isoform H17 of Myeloperoxidase precursor	Myeloperoxidase precursor	[18]
LCP1 plastin-2	L-plastin	[18]
*	Azurocidin	[18]
CAMP antibacterial protein FALL-39 precursor	Antibacterial protein FALL-39	[18]
^†	Gp-340 variant protein	[18]
^†	Similar to mouse von Ebner salivary gland protein, isoform 2	[18,45]
SERPINB1 leukocyte elastase inhibitor	Leukocyte elastase inhibitor	[18]
S100A8 protein S100-A8	Calgranulin A (S100-A8)	[18,80]
S100A9 protein S100-A9	Calgranulin B (S100-A9), calprotectin	[18,77,80]
S100A12 protein S100-A12	Calgranulin C (S100-A12)	[13]
DEFA1; LOC653600; LOC728358 neutrophil defensin 1 precursor	Neutrophil defensins 1-3	[13,18,77]
^†	Bactericidal/permeability-increasing protein (BP1)	[77]
^‡	Insulin-like growth factor binding protein 1 (IGFBP-1) fragments	[18,45]
MMP8 neutrophil collagenase precursor	MMP-8	[81]
MMP9 matrix metalloproteinase-9 precursor	MMP-9	[75]
^‡	MMP-2	[82]
TIMP1 metalloproteinase inhibitor 1 precursor	Metalloproteinase inhibitor 1 (TIMP-1)	[75]
MIF Macrophage migration inhibitory factor	Macrophage migration inhibitory factor	[78]
^†	C-reactive protein (CRP)	[74,79]
LTF growth-inhibiting protein 12	Lactoferrin	[76]

*differentially expressed, but not statistically significant.

^†not observed.

^‡observed, not differentially expressed.

Table III.  Proteins differentially expressed in amniotic fluid samples of patients with preterm delivery with IAI, compared with patients without IAI who delivered at term.

Accession	Gene name, description	Samples
		Protein ID		Ni	No	Differential Expression
		Up	Pp			R	p-value
IPI00007047	S100A8 protein S100-A8	10	1.000	19	17	inf*	4.4 E − 05
IPI00218131	S100A12 protein S100-A12	1	0.610	4	4	Inf*	2.7 E − 02
IPI00027769	ELA2 leukocyte elastase precursor	2	0.998	9	9	27.4	2.0 E − 02
IPI00006988	RETN resistin precursor	1	0.610	15	14	23.5	8.0 E − 04
IPI00180240, IPI00220828, IPI00719405, IPI00815642, IPI00815829, IPI00815835, IPI00816008, IPI00816288, IPI00816464	TMSL3 thymosin-like 3	6	1.000	14	13	12.1	2.0 E − 03
IPI00007244, IPI00236554, IPI00236556	MPO isoform H17 of Myeloperoxidase precursor	28	1.000	18	17	8.2	2.1 E − 03
IPI00005721, IPI00021827	DEFA1; LOC653600; LOC728358 neutrophil defensin 1 precursor	4	1.000	17	16	7.5	5.5 E − 04
IPI00019600	UBE2V2 ubiquitin-conjugating enzyme E2 variant 2	2	0.997	4	4	6.5	2.9 E − 02
IPI00027462	S100A9 protein S100-A9	14	1.000	20	18	5.2	3.8 E − 04
IPI00028064	CTSG cathepsin G precursor	4	1.000	10	10	5.0	4.6 E − 03
IPI00018534, IPI00020101, IPI00152906, IPI00303133, IPI00329665, IPI00419833, IPI00554798, IPI00719084, IPI00794461, IPI00815755	HIST1H2BL histone H2B type 1-L	4	1.000	18	17	4.6	2.5 E − 03
IPI00292532	CAMP antibacterial protein FALL-39 precursor	5	1.000	4	4	4.5	2.9 E − 02
IPI00027509	MMP9 matrix metalloproteinase-9 precursor	9	1.000	17	17	4.5	8.2 E − 04
IPI00019502	MYH9 Myosin-9	8	1.000	8	7	4.5	1.2 E − 03
IPI00171611, IPI00216402, IPI00219038, IPI00465070, IPI00719351	HIST2H3C; HIST2H3A histone H3.2	5	1.000	16	15	4.3	8.4 E − 03
IPI00018873, IPI00472879	PBEF1 isoform 1 of nicotinamide phosphoribosyltransferase	3	1.000	9	8	3.9	1.2 E − 03
IPI00419585	PPIA; LOC653214; LOC654188 peptidyl-prolyl cis-trans isomerase A	8	1.000	19	17	3.8	4.3 E − 05
IPI00465248	ENO1 Isoform α-enolase of α-enolase	7	1.000	13	11	3.7	5.4 E − 03
IPI00759644	FK506 binding protein12	1	0.610	17	15	3.7	7.3 E − 05
IPI00217466	HIST1H1D histone H1.3	1	0.610	6	5	3.5	1.7 E − 02
IPI00021439, IPI00021440	ACTB actin, cytoplasmic 1	16	1.000	21	19	3.5	8.9 E − 06
IPI00220644, IPI00479186	PKM2 isoform M1 of Pyruvate kinase isozymes M1/M2	5	1.000	12	11	3.4	3.9 E − 04
IPI00010471	LCP1 plastin-2	14	1.000	19	17	3.3	3.2 E − 04
IPI00453473	HIST1H4E; HIST1H4F; HIST1H4A; HIST1H4K; HIST1H4C; HIST1H4L; HIST2H4A; HIST1H4D; HIST2H4B; HIST1H4H; HIST1H4B; HIST1H4I; HIST1H4J; HIST4H4 histone H4	7	1.000	17	16	3.2	3.8 E − 03
IPI00012011, IPI00784459	CFL1 cofilin-1	2	0.998	16	14	2.9	2.4 E − 04
IPI00298860	LTF growth-inhibiting protein 12	36	1.000	21	18	2.9	3.2 E − 04
IPI00021085	PGLYRP1 peptidoglycan recognition protein precursor	2	0.998	15	14	2.9	7.8 E − 05
IPI00218834, IPI00640044, IPI00796109	FCGR3A low affinity immunoglobulin-γ-Fc region receptor III-A precursor	3	1.000	18	16	2.9	6.1 E − 04
IPI00013508, IPI00759776	ACTN1 α-actinin-1	3	1.000	4	4	2.8	2.9 E − 02
IPI00178083, IPI00218319, IPI00218320, IPI00382894	TPM3 29 kDa protein	8	1.000	12	11	2.7	1.3 E − 03
IPI00788802	TKT transketolase variant (Fragment)	9	1.000	16	15	2.6	3.4 E − 04
IPI00216318, IPI00640721, IPI00645801, IPI00759832, IPI00795517	YWHAB isoform long of 14-3-3 protein β/α	2	0.610	13	12	2.6	4.0 E − 05
IPI00299547	LCN2 neutrophil gelatinase-associated lipocalin precursor	6	1.000	13	13	2.6	7.0 E − 03
IPI00291005	MDH1 malate dehydrogenase, cytoplasmic	3	1.000	7	7	2.5	4.1 E − 03
IPI00027341	CAPG macrophage-capping protein	5	1.000	19	17	2.5	2.7 E − 04
IPI00217987, IPI00645887	ITGAM integrin α-M precursor	5	1.000	7	6	2.5	1.2 E − 03
IPI00418471, IPI00827679	VIM vimentin	11	1.000	17	15	2.4	4.5 E − 05
IPI00002147	CHI3L1 chitinase-3-like protein 1 precursor	4	1.000	17	16	2.4	5.2 E − 04
IPI00293276, IPI00790382	MIF macrophage migration inhibitory factor	2	0.998	19	17	2.4	2.1 E − 03
IPI00465028, IPI00797687	TPI1 triosephosphate isomerase 1 variant	7	1.000	14	13	2.3	4.2 E − 04
IPI00032292, IPI00642739	TIMP1 Metalloproteinase inhibitor 1 precursor	5	1.000	19	17	2.3	1.6 E − 05
IPI00027444	SERPINB1 leukocyte elastase inhibitor	1	0.610	11	10	2.3	2.3 E − 05
IPI00219757, IPI00796076	GSTP1 glutathione S-transferase P	4	1.000	18	16	2.3	2.3 E − 05
IPI00299024	BASP1 brain acid soluble protein 1	1	0.610	14	13	2.3	1.9 E –03
IPI00021263, IPI00790768	YWHAZ 14-3-3 protein zeta/delta	6	1.000	19	18	2.2	1.6 E − 03
IPI00010270	RAC2 Ras-related C3 botulinum toxin substrate 2 precursor	3	1.000	8	7	2.2	2.1 E − 02
IPI00019755	GSTO1 glutathione transferase omega-1	2	0.998	19	17	2.1	1.7 E − 06
IPI00783881	SFTPA1 similar to Pulmonary surfactant-associated protein A1 precursor	5	1.000	9	8	2.1	3.6 E − 02
IPI00075248, IPI00386621, IPI00794543	CALM2; CALM1; CALM3 calmodulin	2	0.998	17	16	2.1	9.1 E − 04
IPI00807640	HSPA1B; HSPA1A heat shock 70 kDa protein 1B	5	1.000	19	17	2.1	8.5 E − 05
IPI00465439	ALDOA fructose-bisphosphate aldolase A	9	1.000	11	10	2.1	7.9 E − 04
IPI00219525, IPI00747533	PGD 6-phosphogluconate dehydrogenase, decarboxylating	1	0.610	15	14	2.1	5.1 E − 03
IPI00003815, IPI00793767, IPI00794402, IPI00796541	ARHGDIA Rho GDP-dissociation inhibitor 1	1	0.610	14	13	2.1	2.0 E − 04
IPI00033494	MRLC2 myosin regulatory light chain	1	0.610	10	9	2.0	5.7 E − 03
IPI00216298, IPI00552768	TXN thioredoxin	2	0.991	12	11	1.9	4.5 E − 03
IPI00025252, IPI00657680	PDIA3 protein disulfide-isomerase A3 precursor	5	1.000	16	15	1.9	6.4 E − 04
IPI00465436	CAT catalase	7	1.000	16	16	1.9	1.9 E − 03
IPI00219365	MSN moesin	11	1.000	16	15	1.9	7.6 E − 04
IPI00169383	PGK1 phosphoglycerate kinase 1	4	1.000	12	12	1.9	5.0 E − 04
IPI00216691	PFN1 profilin-1	3	1.000	14	13	1.9	1.3 E − 03
IPI00012887, IPI00640540	CTSL1 cathepsin L precursor	1	0.610	19	17	1.9	3.9 E − 03
IPI00003362	HSPA5 HSPA5 protein	8	1.000	16	15	1.8	9.0 E − 04
IPI00453476	hCG_2015269 similar to phosphoglycerate mutase 1 (phosphoglycerate mutase isozyme B) (PGAM-B) (BPG-dependent PGAM 1) isoform 1	6	1.000	15	14	1.8	6.3 E − 04
IPI00413778	FKBP1A FKBP1A protein	1	0.610	8	7	1.8	9.3 E − 03
IPI00013808	ACTN4 α-actinin-4	8	1.000	18	16	1.8	4.4 E − 05
IPI00012048, IPI00026260, IPI00029091, IPI00375531, IPI00604590, IPI00790856, IPI00791971, IPI00795292, IPI00798187	NME1 nucleoside diphosphate kinase A	1	0.610	6	6	1.8	1.3 E − 02
IPI00220642	YWHAG 14-3-3 protein-γ	1	1.000	15	14	1.7	7.0 E − 06
IPI00004573	PIGR polymeric-immunoglobulin receptor precursor	5	1.000	17	15	1.7	3.3 E − 02
IPI00302592, IPI00333541, IPI00644576	FLNA filamin A, α	5	1.000	14	13	1.7	1.0 E − 03
IPI00291175, IPI00307162	VCL isoform 1 of Vinculin	3	1.000	13	12	1.7	1.6 E − 02
IPI00027846	MMP8 neutrophil collagenase precursor	4	1.000	5	5	1.7	7.9 E − 03
IPI00018146, IPI00796727	YWHAQ 14-3-3 protein theta	2	0.610	12	12	1.6	1.2 E − 02
IPI00479145	KRT19 keratin, type I cytoskeletal 19	5	1.000	19	17	1.6	6.0 E − 03
IPI00019038	LYZ lysozyme C precursor	4	1.000	18	16	1.6	2.4 E − 03
IPI00025512	HSPB1 heat-shock protein β-1	2	0.998	10	11	1.6	6.2 E − 03
IPI00000874, IPI00640741, IPI00641244	PRDX1 peroxiredoxin-1	3	0.998	16	16	1.6	3.9 E − 03
IPI00029997	PGLS 6-phosphogluconolactonase	3	1.000	14	12	1.6	2.7 E − 02
IPI00022488	HPX hemopexin precursor	19	1.000	19	18	0.7	1.4 E − 02
IPI00297646	COL1A1 collagen α-1(I) chain precursor	10	1.000	19	17	0.6	2.4 E − 02
IPI00025465, IPI00830128	OGN mimecan precursor	4	1.000	10	9	0.6	1.7 E − 02
IPI00026270	CPM carboxypeptidase M precursor	1	0.610	15	15	0.5	4.4 E − 05
IPI00010341	PRG2 bone-marrow proteoglycan precursor	3	1.000	16	14	0.5	8.3 E − 03
IPI00219832	ABP1 isoform 2 of Amiloride-sensitive amine oxidase [copper-containing] precursor	13	1.000	20	17	0.5	7.7 E − 05

U_p is the number of peptides contributing to protein identification probability (P_p). N_i and N_o are the numbers of samples in which peptides from each protein were observed in patients delivering preterm with IAI and patients with preterm labor who delivered at term, respectively. A relative peptide expression value is determined by normalizing to the reference pool reporter ion intensity. The expression of a protein in a sample relative to the reference pool is determined by computing the median of all relative peptide expression values corresponding to that protein. The median of these individual values is formed for the two groups (N_i and N_o), and their ratio is the protein expression ratio R (values >1.0 correspond to over-expression in the IAI group compared with the term delivery group). p-values were computed with the Mann–Whitney two-sided rank sum test. Protein identities for S100A12, RETN, FK506, HIST1H1D, YWHAB, BASP1, SERPINB1, ARHGDIA, PGD, MRLC2, CTSL1, FKBP1A, NME1, YWHAQ, and CPM were manually validated. Criteria for inclusion in this list were (a) confirmed protein identity, (b) p-value for differential expression of the protein of less than 0.05, and (c) the fold-change between groups of at least 1.5.

*The median value among the term delivery group was zero, whereas the median value in the IAI group was finite.

Table IV.  Proteins differentially expressed in amniotic fluid samples of patients with preterm delivery without IAI, compared with patients who delivered at term.

Accession	Gene name, description	Samples
		Protein ID		Ni	No	Differential Expression
		Up	Pp			R	p-value
IPI00006988	RETN Resistin precursor	1	0.610	15	14	5.3	0.039
IPI00180240, IPI00220828, IPI00719405, IPI00815642, IPI00815829, IPI00815835, IPI00816008, IPI00816288, IPI00816464	TMSL3 thymosin-like 3	6	1.000	14	13	2.6	0.005
IPI00008580	SLPI Antileukoproteinase precursor	2	0.998	8	7	2.1	0.040
IPI00298860	LTF Growth-inhibiting protein 12	36	1.000	20	18	1.9	0.027
IPI00220249, IPI00410152, IPI00784258	LTBP1 Latent-transforming growth factor β-binding protein, isoform 1L precursor	2	0.998	4	4	0.6	0.029
IPI00025465, IPI00830128	OGN Mimecan precursor	4	1.000	10	9	0.6	0.025

Headings, calculations, and criteria for inclusion are as described for Table III.

Figure 2.  A description of the gene ontology biological processes over-represented in the amniotic fluid of women with spontaneous preterm labor in the presence of intra-amniotic infection/inflammation. (A) Pie chart that displays the main four gene ontology terms that were significantly enriched in the amniotic fluid of women with preterm labor with IAI: (1) metabolism/catabolism; (2) mobility, localization and targeting; (3) anti-apoptosis; and (4) host defense. (B) The four main branches in the network diagram represent four gene ontology terms described in the pie chart. Color nodes represent the biological processes that are significantly over-represented (the darker the color, the greater the significance is). The open nodes represent biological processes that did not reach statistical significance. As described in detail in the text, BiNGO (version 2.0) was used to calculate GO term enrichment, and CytoScape (version 2.5.1) was used to visualize the resulting network of GO biological processes.

Figure 3.  Clusters of biological processes identified in the main four gene ontology terms. The four pie charts represent the main gene ontology terms over-represented in the amniotic fluid of women with spontaneous preterm labor in the presence of intra-amniotic infection/inflammation. Each pie chart displays clusters of biological processes that are significantly enriched and functionally inter-related.

Gravett MG, Novy MJ, Rosenfeld RG, Reddy AP, Jacob T, Turner M, McCormack A, Lapidus JA, Hitti J, Eschenbach DA, et al Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers. JAMA 2004;292:462–469.

 PubMed Web of Science ®Google Scholar

Bujold E, Romero R, Kusanovic JP, Erez O, Gotsch F, Chaiworapongsa T, Gomez R, Espinoza J, Vaisbuch E, Mee KY, et al Proteomic profiling of amniotic fluid in preterm labor using two-dimensional liquid separation and mass spectrometry. J Matern Fetal Neonatal Med 2008;21:697–713.

 PubMed Web of Science ®Google Scholar

Park SJ, Yoon WG, Song JS, Jung HS, Kim CJ, Oh SY, Yoon BH, Jung G, Kim HJ, Nirasawa T. Proteome analysis of human amnion and amniotic fluid by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomics 2006;6:349–363.

 PubMed Web of Science ®Google Scholar

Espinoza J, Chaiworapongsa T, Romero R, Edwin S, Rathnasabapathy C, Gomez R, Bujold E, Camacho N, Kim YM, Hassan S, et al Antimicrobial peptides in amniotic fluid: defensins, calprotectin and bacterial/permeability-increasing protein in patients with microbial invasion of the amniotic cavity, intra-amniotic inflammation, preterm labor and premature rupture of membranes. J Matern Fetal Neonatal Med 2003;13:2–21.

 PubMedGoogle Scholar

Buhimschi I, Christner R, Buhimschi C, Chaiworapongsa T, Romero R. Proteomic analysis of preterm parturition: a novel method of identifying the patient at risk for preterm delivery. Am J Obstet Gynecol 2002;187:S55.

 Google Scholar

Nien JK, Yoon BH, Espinoza J, Kusanovic JP, Erez O, Soto E, Richani K, Gomez R, Hassan S, Mazor M, et al A rapid MMP-8 bedside test for the detection of intra-amniotic inflammation identifies patients at risk for imminent preterm delivery. Am J Obstet Gynecol 2006;195:1025–1030.

 PubMed Web of Science ®Google Scholar

Athayde N, Romero R, Gomez R, Maymon E, Pacora P, Mazor M, Yoon BH, Fortunato S, Menon R, Ghezzi F, et al Matrix metalloproteinases-9 in preterm and term human parturition. J Matern Fetal Med 1999;8:213–219.

 PubMedGoogle Scholar

Peltier MR, Barney BM, Brown MB. Effect of experimental genital mycoplasmosis on production of matrix metalloproteinases in membranes and amniotic fluid of Sprague-Dawley rats. Am J Reprod Immunol 2007;57:116–121.

 PubMed Web of Science ®Google Scholar

Chaiworapongsa T, Romero R, Espinoza J, Kim YM, Edwin S, Bujold E, Gomez R, Kuivaniemi H. Macrophage migration inhibitory factor in patients with preterm parturition and microbial invasion of the amniotic cavity. J Matern Fetal Neonatal Med 2005;18:405–416.

 PubMed Web of Science ®Google Scholar

Yoon BH, Jun JK, Park KH, Syn HC, Gomez R, Romero R. Serum C-reactive protein, white blood cell count, and amniotic fluid white blood cell count in women with preterm premature rupture of membranes. Obstet Gynecol 1996;88:1034–1040.

 PubMed Web of Science ®Google Scholar

Shim SS, Romero R, Jun JK, Moon KC, Kim G, Yoon BH. C-reactive protein concentration in vaginal fluid as a marker for intra-amniotic inflammation/infection in preterm premature rupture of membranes. J Matern Fetal Neonatal Med 2005;18:417–422.

 PubMed Web of Science ®Google Scholar

Pacora P, Maymon E, Gervasi MT, Gomez R, Edwin SS, Yoon BH, Romero R. Lactoferrin in intrauterine infection, human parturition, and rupture of fetal membranes. Am J Obstet Gynecol 2000;183:904–910.

 PubMed Web of Science ®Google Scholar