Shotgun proteomic analysis of vaginal fluid from women in late pregnancy

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Reproductive Sciences

DOI: 10.1177/1933719107311189 2008; 15; 263 Reproductive Sciences

Laura L. Klein, Karen R. Jonscher, Margaret J. Heerwagen, Ronald S. Gibbs and James L. McManaman Shotgun Proteomic Analysis of Vaginal Fluid From Women in Late Pregnancy

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Shotgun Proteomic Analysis of Vaginal FluidFrom Women in Late Pregnancy

Laura L. Klein, MD, Karen R. Jonscher, PhD, Margaret J. Heerwagen, BS,Ronald S. Gibbs, MD, and James L. McManaman, PhD

�Liquid chromatography and tandem mass spectrometry without prior fractionation (shotgun proteomics)were used to analyze vaginal fluid from patients admitted for signs and symptoms of preterm labor. Thepatients had an average age of 26.3 ± 5.9 years, a gestational age of 30.5 ± 2.5 weeks, and a mediancervical dilation of 1 cm (range, 0-6 cm). None of the patients exhibited signs of vaginal infection atthe time of enrollment. Shotgun proteomics yielded reproducible identifications ( R = 0.973) of morethan 40 proteins in vaginal fluid samples, such as plasma proteins, epithelial structural proteins, andseveral immunoregulatory proteins, including some that were previously linked to intra-amniotic infec-tion. This initial characterization of the vaginal fluid proteome using a nonbiased, high-throughputtechnique yields reproducible results in late pregnancy. The presence of host defense proteins in vaginalfluid suggests that this technique may be useful for future study of inflammation-related preterm birth.

�KEY WORDS: Proteomics, preterm labor, vaginal fluid, antimicrobial,immunomodulatory, chemotaxis.

particularly at less than 30 weeks of gestation.2,3 Althoughoccasionally systemic infections such as Listeriosis causepreterm birth via hematogenous spread to the uterus, byfar, the most common mechanism of infection-relatedpreterm birth is ascending infection from the lower gen-ital tract. This lower genital tract infection can occurweeks to months prior to the clinical onset of pretermlabor, as evidenced by the strong association of bacterialvaginosis prior to 16 weeks of gestation with pretermbirth.4 Mucous secretions in the genital tract are knownto contain antimicrobial and antiviral substances thatrestrict the growth of infectious organisms,5 which raisesthe possibility that premature birth due to ascendinginfections is, in part, due to impairment of the genitaltract’s innate defense mechanisms. At present, few detailsexist about the molecules or the nature of the mecha-nisms that make up this defense.

Proteomic techniques allow characterization of theglobal expression of proteins in biologic samples; a surface-enhanced laser desorption ionization (SELDI) pro-teomics approach has recently been used to demonstrateelevated expression of certain immunoregulatory pro-teins in amniotic fluid samples from nonhuman primateswith intra-amniotic infections and in a cohort of womenwith subclinical intra-amniotic infection and preterm

The current rate of preterm birth in the UnitedStates is approximately 12.5%, which represents asteady increase over the past 20 years.1 Prematurity

accounts for a large percentage of neonatal mortality innonanomalous infants and can be associated with lifelongdisability in survivors.2 Infection and inflammation havebeen demonstrated to play key roles in preterm labor,

From the Departments of Obstetrics and Gynecology (LLK, RSG, JLM);Anesthesiology, Clinical Nutrition Research Unit (KRJ); Physiology andBiophysics (JLM); and the Graduate Program in Reproductive Sciences (MJH,JLM), University of Colorado at Denver & Health Sciences Center, Aurora,Colorado. LLK is currently at Memorial Hospital Pikes Peak Maternal FetalMedicine Center, Colorado Springs, Colorado. LLK and KRJ contributedequally to this work.

Supported by grants from the March of Dimes (JLM, RSG); University ofColorado at Denver & Health Sciences Center, Department of Obstetrics andGynecology, Academic Enrichment Fund (LLK and JLM); and NationalInstitutes of Health (NIH) P30 DK048520 (Mass Spectrometry Core). Theauthors thank Eric J. Kushner for technical assistance with 2D gels and Dr M.C. Neville for helpful comments.The CNRU Mass Spectrometry Core is sup-ported by NIH/NIDDK P30 DK048520.

Address correspondence to: James L. McManaman, PhD, Division ofReproductive Sciences, Mail Stop 8309, University of Colorado Denver HealthSciences Center,Aurora, CO 80045. E-mail: [email protected]

Reproductive SciencesVol. 15 No. 3 March 2008 263-273DOI. 10.1177/1933719107311189© 2008 by the Society for Gynecologic Investigation

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contractions.6 We have used SELDI-based proteomicspreviously to reliably distinguish infected from unin-fected animals in a rabbit model of intrauterine infec-tion.7 This technique has also been used to establishan amniotic fluid protein profile in women with intra-amniotic infection that accurately predicted imminentpreterm delivery8 and to identify differences in the pro-tein profiles of vaginal fluid9 or aminotic fluid-vaginalfluid mixtures10 between women with and without intra-amniotic infection. Although these results indicate thegeneral importance of proteomic approaches to the iden-tification molecules associated with clinical infections ofthe reproductive tract and preterm birth, in each of thesestudies, additional purification and mass spectrometryprocedures were required to obtain protein identities, andonly a limited number of proteins were ultimately iden-tified. Thus, establishment of more sensitive and directprotein identification procedures may improve the abilityto identify potential markers of preterm birth.

Although the amniotic cavity is a logical site forevaluation of the preterm labor process, amniocentesis isan invasive procedure that carries a small but known riskto the pregnancy.Vaginal fluid, however, can be samplednoninvasively and monitored serially during a pregnancyat high risk of preterm birth and other adverse outcomes.In this study, we use a high-throughput shotgun pro-teomics method11,12 for direct identification of proteinsin vaginal and amniotic fluids.This method has been usedto obtain an initial vaginal fluid proteome of womenadmitted in late pregnancy with signs and symptoms ofpreterm labor. Our data demonstrate that multipleimmunomodulatory and antimicrobial proteins, includ-ing those previously associated with intra-amniotic infec-tion, are present in vaginal fluid of women with pretermcontractions but without infection or inflammation.

MATERIALS AND METHODS

Patient Population and Sample Collection

The study protocol was approved by the Colorado MultipleInstitutional Review Board (protocol no. 05-0622).Patients presenting to the University of Colorado Hospitalat 20 to 34 weeks of gestation with complaints of con-tractions were eligible for study participation.Those withsignificant vaginal bleeding, rupture of membranes, or ahistory of sexual intercourse within 24 hours wereexcluded, as the vaginal proteome would be expected to

be altered in these patients. Study patients underwent asterile speculum examination, at which time vaginal fluidsamples were collected for proteomic analysis. Samplescollected for 2-dimensional gel electrophoresis wereobtained using a vaginal wash technique, in which 3 mLof sterile saline was instilled into the posterior vaginalfornix, agitated, and then reaspirated. Samples collectedfor shotgun proteomics analysis were obtained by saturat-ing Dacron swabs with secretions from the posterior vagi-nal fornix and immediately placing them into 1 mL ofpreservative solution (0.5% formic acid/5 % acetonitrile).All samples were centrifuged at 1800 rpm for 5 minutesat room temperature within 30 minutes of collection.Thesupernatants were aliquoted and frozen at –70°C.

All study patients were evaluated for vaginal infec-tions using wet mount, pH, and amine odor tests. Patientmanagement was otherwise not altered by study partici-pation. Patients were treated as clinically indicated. Ifamniocentesis was performed for clinical indications, 2mL of amniotic fluid was reserved for proteomic analysis.In such patients, vaginal fluid was sampled prior toamniocentesis. Medical record review was conducted todetermine delivery and neonatal outcomes.

Shotgun ProteomicAnalysis of Vaginal Proteins

Proteins in vaginal fluid supernatants were precipitated(2D Clean Up Kit; GE Healthcare, Piscataway, NJ),reconstituted in 8M urea, and digested overnight withtrypsin following reduction (50 mM dithiothreitol) andalkylation (1 M iodoacetamide). Digests were stoppedwith 1 μL of formic acid and were stored at –20°C priorto analysis. Nanoscale liquid chromatography and tandemmass spectrometry (LC/MS/MS), or shotgun pro-teomics, was used to analyze the digests. Briefly, aliquotsof the digest corresponding to 10% to 20% of total vol-ume were loaded (5 minutes, 15 μL/min) onto ananospray column (75 μm ID × 10 cm) using a micro-well plate autosampler. Peptides were eluted into the massspectrometer using an Agilent 1100 HPLC and a gradi-ent of increasing buffer B (90% ACN, 0.1% formic acid)at a flow rate of 300 nL/min. Buffer A was 0.1% formicacid.The gradient was ramped from 3% to 20% over 20minutes, then from 20% to 55% over 35 minutes, andfrom 55% to 90% over 40 minutes. Finally, buffer B washeld at 90% for 10 minutes and then returned to initialconditions (3%) and equilibrated for 30 minutes. Spectra

264 Reproductive Sciences Vol. 15, No. 3, March 2008 Klein et al



were collected over an m/z range of 350 to 2000 Da onan Agilent LC/MSD Trap XCT Plus mass spectrometer.Three MS/MS spectra were collected for the 3 mostabundant m/z values; those masses were then excludedfrom analysis for 30 to 120 seconds, and the next 3 mostabundant m/z values were selected for fragmentation toenable analysis of lower abundance peptide ions.

Protein IdentificationUsing Database Searching

Proteins were identified by searching the NCBInr,SwissProt, and IPI databases using the SpectrumMillProteomics Workbench (Agilent Technologies, SantaClara, CA). Parameters used in the database search are asfollows: monoisotopic mass, peptide mass tolerance of 1.5Da, fragment ion mass tolerance of 0.7 Da, tryptic pep-tides allowing for only 2 missed cleavages, and car-bamidomethylation of Cys as a fixed modification.Database matches were validated by reverse databasescoring using SpectrumMill software. Proteins were char-acterized based on their SpectrumMill protein scores(MS scores). The MS score is a composite value thatreflects the number of unique identifying peptides andthe relative spectral intensity of each peptide and thusprovides an estimate of relative abundance of each iden-tified protein. Proteins with MS scores greater than 13,peptide scores greater than 10, and scored percentageintensity of 70% were used as a cutoff for initial hit vali-dation. Each identified protein required validation of atleast 2 unique peptides.The NCBInr database contained133 405 sequences (5/1/05 release) with the humantaxonomy filter, SwissProt had 12 231 sequences (5/10/05release) with the human taxonomy filter, and IPI_HUMAN had 57 032 sequences (12/6/05 release).

Two-Dimensional Gel Electrophoresis

Supernatants were vacuum concentrated from about 1.0mL to approximately 300 μL. Concentrated samples (∼50μL) were buffer exchanged using a Zeba Desalt SpinColumn (0.5 mL; Pierce Biotechnology, Rockford, IL)into chaotropic lysis buffer (7M urea, 2M thiourea, 4%CHAPS, 50 mM DTT, 0.2% pH 3-10 carrierampholytes, 1% protease inhibitor mix). Samples werediluted to 200 μL by addition of chaotropic lysis bufferplus 0.5 μL of 1% bromophenol blue and applied to 11cm pH 3-10 IPG strips (Bio-Rad Laboratories, Hercules,

CA). After active hydration (50 V, 20°C, 12 hours), theproteins were separated for 30 000 volt-hours using aProtean IEF cell (Bio-Rad Laboratories) with a voltagegradient ending at 6 kV. Following isoelectric focusing,the strips were first equilibrated with 10 mg/mL dithio-threitol in equilibration buffer (20% glycerol, 20%sodium dodecyl sulfate [SDS], 1.5 M Tris pH 8.8, and 6M urea) and then with 25 mg/mL iodoacetamide inequilibration buffer.

The second dimension of separation was carried outusing a Criterion electrophoresis chamber or Dodeca cellwith 10% to 20% acrylamide precast gels (Bio-RadLaboratories). Equilibrated strips were cemented ontoSDS polyacrylamide gel electrophoresis gels with 0.6%agarose. Proteins were separated by molecular weightusing 180 V for approximately 1 hour. Gels were main-tained at 15°C during electrophoresis. Proteins werestained overnight with Sypro Ruby Red (Bio-RadLaboratories) and imaged using a 9600 Typhoon TrioFluorescence Imager (GE Healthcare) at a 200-μm reso-lution. Molecular weight, isolecteric points, and spotalignments were determined using feature comparisonfunctions of ImageMaster software (GE Healthcare).

Statistical Analyses

Correlation analysis (Pearson) was used to determine theinterassay relationship between populations of identifiedproteins. A highly positive correlation (R = 0.973) wasobserved between MS scores of individual proteins in quad-ruplicate assays of the same sample. The relative interassayvariability of MS scores for each protein was calculated usingthe following equation: (SD/MS score) × 100.

RESULTS

Patient Population

Vaginal fluid samples were collected from 7 patients withthe following characteristics: maternal age (mean ± SD) =27.9 ± 6.7 years, gestational age = 30.1 ± 2.3 years, andmedian cervical dilation = 1 cm (range, 0-6 cm). None ofthe patients were found to have any vaginal infection at thetime of enrollment.All 7 patients were admitted and treatedfor a diagnosis of preterm labor and received tocolytics.The patients had the following clinical characteristics:2 were positive for fetal fibronectin, 2 delivered before 34weeks (27 and 33 weeks), and 5 delivered at or near term

Proteomic Analysis of Human Vaginal Fluid Reproductive Sciences Vol. 15, No. 3, March 2008 265



(≥34 weeks). None of the subjects had symptoms of clini-cal chorioamnionitis at the time of sample collection.

Two-Dimensional Gel Electrophoresis

Preliminary 2-dimensional (2D) gel electrophoresisanalysis revealed that vaginal fluid contained relativelyfew proteins (Figure 1). Overall, vaginal fluid samplesappeared to have similar 2D gel electrophoresis profiles,although some intersample variability in spot intensitywas observed for certain proteins (Figure 1). However, wewere unable to perform a quantitative differential analy-sis of vaginal fluid protein expression using this techniquebecause the protein concentrations of vaginal fluid werefrequently below detection limits of the Bradford assay(data not shown). In addition, 2D gel electrophoresis wastime-consuming and labor intensive, which limits its use-fulness as a high-throughput proteomic screening proce-dure for vaginal fluid.

Shotgun Proteomics

The relatively simple protein composition of vaginal fluidobserved in 2D gels suggested that it might be possible todirectly identify vaginal fluid proteins by shotgun pro-teomics11,12 without the need for prior purification by2D gels or other methods.6,7,9 In this approach, the pro-tein mixture is digested and the resultant peptides identi-fied using either single or multidimensional LC/MS/MS.Protein identification is then accomplished via mappingthe peptides to a protein locus.To assess the feasibility ofshotgun proteomics for identifying vaginal fluid proteins,a single sample of unfractionated vaginal fluid collectedusing a Dacron swab was digested with trypsin and sub-jected to repeated (N = 4) analyses by shotgun pro-teomics. Figure 2A shows that there was a high degree ofreproducibility in the retention times and signal inten-sities of peptide ions between LC/MS/MS runs. Wedetermined the relative interassay variability of theSpectrumMill MS score of each identified protein as ameans of estimating the accuracy and reliability of pep-tide identifications obtained by our method.The relativeinterassay variability in protein identification was lessthan 15% for proteins in which at least 2 unique peptideswere identified (MS scores ≥20).The percentage of pro-teins with MS scores ≥20 was 69% ± 4% (SEM) of thetotal number of identified proteins. Thus, identificationsappear to be reliable for most of the proteins identified byshotgun proteomics.

Partial Human Vaginal Fluid Proteome

We analyzed the protein compositions of vaginal fluidfrom 8 patients who presented with premature contrac-tions by shotgun proteomics.Vaginal fluid from 1 patientyielded no interpretable results because of contaminationof the gel used in transvaginal ultrasound prior to samplecollection. Subsequent analyses were limited to samplesfrom patients who had not received transvaginal ultra-sound or a vaginal examination in the prior 12 hours.This included 5 patients who delivered at or near termand 2 patients who delivered preterm (<34 weeks). Usingan MS score cutoff of 20, we identified 39 proteins withaverage MS scores ≥20 (Table 1) from these 7 patients.Most of the identified proteins were serum or epithelialproteins. However, several host defense proteins andimmune modulatory proteins also were identified.Amnioticfluid samples were obtained for clinical indications from


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Figure 1. Two-dimensional (2D) gel analyses of vaginal fluid sam-ples from 2 patients admitted with signs and symptoms of pretermlabor that subsequently delivered at or near term. (A) Patient admit-ted at 33 weeks of gestation and released without tocolysis. (B) Patientadmitted at 33 weeks of gestation. She was positive for fetal fibronectinand received tocolysis for 24 hours and was then released. Circledspots in B indicate proteins not found in A.Arrows indicate represen-tative spots that are shared by both patients. Polypeptide pattern wasvisualized by Sypro Red staining and fluorescence imaging. Spotswere aligned by computer using feature comparison functions ofImageMaster software.



2 subjects, 1 of whom delivered preterm. From thesesamples, we identified 17 proteins that were common toboth patients; 5 of these also were found in vaginal fluid(Table 2).

Plotted in Figure 3 are fragmentation spectra fromrepresentative peptides used to identify the proteinscalgranulin (S100 A9,Figure 3A) and neutrophil gelatinase–associated lipocalin precursor (NGAL; Figure 3B). Thetheoretical sequence for each peptide is shown above thecorresponding mass spectrum, and the expected mass-to-charge ratios for the b- and y-type ions are listed. TheMS score for the calgranulin-derived peptide, NIETI-INTFHQYSVK, was 18.93. The spectrum was alsosearched against a reversed database, and the differencebetween the reverse and forward scores was 6.38, furtheraiding in the validation of this assignment. Signals weredetected for y3-12 and are displayed in bold and under-lined. Signals that were observed from complementary b

ions included b10-14, as well as water losses from b4 andb5. Peaks labeled with an asterisk indicate loss of waterfrom an expected fragment ion. The significant waterlosses observed correlate well with the presence of mul-tiple S/T residues. The observation of doubly chargedy12 and y13 is consistent with the presence of histidine.

A similar analysis was performed for the peptideSYPGLTSYLVR from NGAL (MS score of 16.22).Thedominant ion in this spectrum resulted from cleavage atthe proline residue. Ions from subsequent internal cleav-ages were also observed and are labeled.The abundancescale has been expanded by a factor of 100 to observe thelow abundance fragment ions. More than 90% of theamino acid residues were observed in the fragmentationspectrum.Again, most signals resulted from y-type cleav-ages (y1-9); however, several complementary b (b2, b3,b6, b7, b10, and b11) and internal (PGL, PGLT, PGLTS,and PGLTSY) fragment ions were also observed.


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Figure 2. Reproducibility of shotgun proteomics. (A) Overlay of repeated tandem mass spectrometry (MS/MS) analyses (N = 4) of a singlehuman vaginal fluid sample, showing close overlap of signal intensities and peak retention times. (B) The relationship between MS scores andtheir relative interassay variability. Proteins with MS scores of 20 or greater have an interassay variability of less than 15%.



DISCUSSION

Previous Studies of VaginalFluid Proteins

Proteins found in the vagina of a pregnant individual mayoriginate from several sources: (1) a transudate of plasma

across the vaginal epithelium; (2) secretion by vaginal orcervical epithelial cells; (3) secretion from immune cellspresent in the vagina such as neutrophils and macrophages;(4) secretion from cells in the upper genital tract, includ-ing the chorion and deciduas; (5) sloughed epithelialcells; and (6) production by organisms in the vaginalflora. Antibody-based tests have been used in previous


Table 1. Proteins Identified in Vaginal Fluid of Women Admitted With Signs and Symptoms of Preterm Labor

Class Molecular Weight Positive Samples Swiss-Prot No.

Cellular proteinsSmall proline-rich protein 3 18 154 6 Q9UBC9Squamous cell carcinoma antigen 1 44 565 5 P29508Cytokeratin-6c 60 069 6 P48666Cytokeratin-13 49 587 5 P13646Involucrin 68 469 5 P07476Epidermal, fatty acid–binding protein 15 033 4 Q01469Cytokeratin-1 65 887 5 P04264Myeloperoxidase precursor 83 869 3 P05164Cytokeratin-4 57 286 4 P19013Polymeric-immunoglobulin receptor precursor (PIGR) 83 314 3 P01833Small proline-rich protein 2A 7966 4 P35326Annexin A2 38 473 3 P07355Histone H4 11 236 3 P62805L-plastin 70 290 2 P1379628-kDa heat-shock protein 22 783 4 P04792Small proline-rich protein IA 9883 6 P35321Glyceraldehyde-3-phosphate dehydrogenase 35 922 4 P04406

Immunoglobulin relatedIg γ-q chain C region 36 106 5 P01857Ig κ chain C region 11 609 5 P01834Ig λ chain C regions 11 237 4 P01842

Serum proteinsSerum albumin precursor 69 367 6 P02768Hemoglobin β subunit 15 867 2 P68871Hemoglobin α subunit 15 126 2 P69905

Protease relatedSquamous cell carcinoma antigen 1 44 565 5 P29508Leukocyte elastase inhibitor 42 742 5 P30740α-1-antitrypsin precursor 46 737 3 P01009Secretory leukocyte protease inhibitor 14 326 6 P03973Cathepsin G precursor 28 837 4 P08311α-1-antichymotrypsin precursor 47 651 2 P01011Leukocyte elastase precursor 28 518 3 P08246

Host defense relatedLactotransferrin precursor 78 339 4 P02788Neutrophil gelatinase–associated lipocalin precursor 22 588 5 P80188Calgranulin B (S100A9) 13 242 6 P06702Transferrin 77 050 3 P02787Annexin A1 38 583 4 P04083Von Ebner minor salivary gland protein 52 442 4 Q8TDL5Neuroblast differentiation-associated protein AHNAK 312 495 3 Q09666Calgranulin A (S100A8) 10 835 6 P05109Lysozyme C precursor 16 537 4 P61626



studies to identify specific proteins in human vaginalfluid; however, to our knowledge, the entire vaginal pro-teome has not been investigated in a nonbiased fashionusing modern high-throughput technologies. In non-pregnant women, the vagina has been found to containserum proteins (albumin, transferrin, immunoglobulins,ceruloplasmin),13 antimicrobial proteins and immune fac-tors (calprotectin, lysozyme, lactoferrin, secretory leuko-protease inhibitor, human β defensins, human neutrophilpeptide/α defensins),14 and interleukins (IL-6, -8, -10,and -12).15

Certain vaginal proteins have been shown previouslyto have potential diagnostic and/or prognostic value withrespect to preterm birth. In the setting of preterm pre-mature rupture of membranes, cervical IL-6 levels havebeen found to be elevated in patients with intra-amnioticinfection16,17 and in those who delivered within 7 days.16

In the setting of preterm labor with intact membranes,intra-amniotic infection has been associated with ele-vated cervical levels of IL-6, IL-1β, IL-1RA, tumornecrosis factor–α,18 IL-8,19 and insulin-like growth factorbinding protein 1.20 Vaginal IL-8 levels have been foundto be elevated in both term and preterm labor and tocorrelate with cervical dilation21 and the presence ofintra-amniotic infection.22 Elevated vaginal fluid concen-trations of neutrophil defensins in the second trimesterhave been associated with increased risk of early pretermbirth (<32 weeks).23 Unfortunately, none of these individual

proteins have been found to have sufficient positive or neg-ative predictive value for clinical use.Vaginal fetal fibronectinhas been demonstrated to have excellent negative predictivevalue; it has a role in ruling out preterm birth.24 However,its positive predictive value is poor, both for preterm birthand for the identification of intra-amniotic infection.25

Vaginal Fluid Proteins Identifiedby Shotgun Proteomics

We found shotgun proteomics to be an efficient andreproducible method for identifying proteins in humanvaginal fluid samples. However, it is clear from these initialanalyses that we have identified only the most abundantvaginal fluid proteins. Some proteins that are commonlyidentified in vaginal secretions using immunoassays—notably, the interleukins—were not identified in our study,


Table 2. Proteins Identified in Amniotic Fluid of PatientsAdmitted for Preterm Contractions

Proteins detected in amniotic and vaginal fluidsSerum albumin precursorSerotransferrin precursorα-1-antitrypsin precursorα-1 antichymotrypsin precursorImmunoglobulins

Proteins detected in amniotic fluid onlyFibronectin precursorCeruloplasmin precursorApolipoprotein-A1 precursorKeratan sulfate proteoglycan lumicanα-1-acid glycoprotein 1 precursor (orosomucoid-1)Vitamin D–binding protein precursorInsulin-like growth factor binding protein 1 precursorComplement C3 precursorAngiotensinogen precursorAMBP protein precursorα-1-B glycoprotein precursorTransthyretin precursor (prealbumin)

Figure 3. Protein identification from peptide fragmentation prod-ucts.Tandem mass spectrometry (MS/MS) spectra from (A) calgran-ulin A and (B) neutrophil gelatinase–associated lipocalin precursor(NGAL) were searched against the SwissProt database usingSpectrumMill. The theoretical peptide sequence best matching theproduct ions was used to generate a list of expected m/z values for theproduct ions.Values for expected ions that were observed in the spec-tra are presented in bold.An asterisk indicates that we did not observea particular fragment ion but did detect its loss of water. Proteins wereidentified by at least 2 peptide hits.



presumably because their concentrations were below thesensitivity limits of our MS technique. In preliminary stud-ies, we found that by using limited mass range acquisition–also termed gas phase fractionation26—we were able toincrease the number of peptides identified by shotgun pro-teomics by approximately 3-fold (data not shown).Thus, itmay be possible to identify less abundant proteins by sim-ply limiting the mass range during MS analysis. In addi-tion, prefractionation by ion exchange and reverse-phasechromatography prior to mass spectrometry27 is likely tofurther improve identification of low-abundance proteins.We are currently evaluating the usefulness of these andother modifications in the identification of low-abundanceproteins in human vaginal fluid using a larger cohort thanthat employed in these initial studies.

Preliminary Characterization ofthe Human Vaginal Proteome

As expected, subsets of plasma and epithelial cell proteinswere identified in human vaginal fluid by shotgun pro-teomics. Of greater interest, however, was the identifica-tion of multiple immunomodulatory and antimicrobialproteins in vaginal fluid samples from most patients.Thepresence of these proteins does not appear to be the resultof inflammatory responses, as none of the patients in ourstudy exhibited evidence of vaginal infection or inflam-mation at the time of enrollment.Whether these proteinsare normal components of human vaginal fluid dur-ing pregnancy or are somehow linked to the prematurecontractions that brought these women into the hospitalis uncertain. However, they do not appear to be specifi-cally linked to preterm birth since they were found invaginal fluid from women who delivered at term as wellas those who delivered preterm.

Among the abundant proteins found in human vagi-nal fluid samples were several inhibitors of neutrophilelastase activity, including leukocyte elastase inhibitor,α-1-antitrypsin, α-1-antichymotrypsin, and secretoryleukocyte protease inhibitor (SLPI). Elastase inhibitoryactivity has been implicated in the protection of epithe-lial barriers from damaging effects of elastases released inresponse to infection.28-30 The presence of multiple elas-tase inhibitors in vaginal fluid of pregnant women sug-gests that redundant protection mechanisms exist withinthe genital tract to maintain epithelial barrier integrity inthe event of infection or inflammation.This possibility issupported by the studies of Draper et al,31 showing thatvaginal fluid SLPI levels are reduced in subjects infected

with Trichomonas vaginalis and that proteases secreted byT vaginalis produce enzymes capable of degrading SLPI.α-1-antitrypsin and α-1-antichymotrypsin are found inserum, and their concentrations increase following acti-vation of acute-phase responses.28,32 However, these fac-tors can also be produced locally by epithelial cells.32

Mucosal epithelial cells, including the cervical mucosa,are also known to express SLPI.33,34 Therefore, it is likelythat antielastase inhibitors in vaginal fluid are derived, inpart, from local production by cervicovaginal epithelialcells. α-1-antitrypsin and α-1-chymotrypsin inhibitorswere also detected in amniotic fluid by shotgun pro-teomics, which confirms previous results based onenzyme activity measurements and 2-dimensional immu-noelectrophoresis.35,36 Although direct transfer of theseproteins into amniotic fluid has not been demonstrated,it is likely that they are derived from maternal blood.36

Several factors linked to host defense mechanismswere also identified in vaginal fluid by shotgun pro-teomics, including antimicrobial proteins (lysozyme c,lactoferrin, transferrin, NGAL, and von Ebner’s minorsalivary gland protein) and immunomodulatory proteins(annexin A1 and calgranulins A and B). The identifica-tion of multiple antimicrobial proteins in vaginal fluid isconsistent with the known importance of mucosal secre-tions in protecting epithelial surfaces from bacterialinfection. However, of particular relevance to the humanfemale genital tract is the presence of lactoferrin, trans-ferring, and NGAL. Lactoferrin and transferrin are iron-binding proteins that inhibit bacterial growth byreducing iron availability.37 Both proteins have beendetected previously in vaginal fluid and are thought tobe a first line of defense against bacterial infection of thegenital tract. Many types of bacteria, however, are able toovercome the effects of lactoferrin and transferrin byproducing low-molecular-weight iron chelators (sider-phores) that can remove iron from these proteins.38 Recentstudies have shown that NGAL is able to avidly bind thesiderphore enterobactin and interfere with enterobacte-rial growth by inhibiting siderophore-mediated ironuptake.39 The presence of NGAL in human intra-amni-otic fluid has been linked to infections associated withpreterm birth.6 Our data, however, show that NGAL ispresent in uninfected human cervical secretions ofwomen during pregnancy and confirm previousimmunoblot data suggesting that NGAL was a compo-nent of human cervical fluid.40

The origin of these proteins in vaginal fluid mayinvolve both transudation and local secretion. Lactoferrin




is secreted by many types of exocrine cells, includingthose of the genital tract.37,41 Although transferrin is themajor iron-transporting protein of blood, there is also evi-dence that it can be synthesized by epithelial cells.42

NGAL was originally isolated from neutrophils,43 but italso is expressed in differentiating epidermal cells44 andgastrointestinal epithelial cells in response to inflammatorysignals or neoplasia.45,46 Because we did not detect signif-icant numbers of neutrophils in vaginal fluid samples frompatients in our study (data not shown), it is likely thatNGAL originated from genital tract epithelial cells, butadditional work will be required to confirm this origin.The presence of multiple proteins that interfere with bac-terial iron metabolism in vaginal fluid suggests that over-lapping mechanisms exist within the genital tract duringpregnancy to inhibit iron-dependent growth of poten-tially harmful bacteria. We also identified von Ebner’sminor salivary gland protein (VEMSGP) in human vagi-nal fluid. Although VEMSGP has not been directlydemonstrated to possess antimicrobial activity, it is struc-turally related to bactericidal permeability-increasing pro-tein (BPI).47 Elevated amniotic fluid levels of BPI areassociated with intra-amniotic infection, preterm birth,and preterm premature rupture of membranes.48 Thus, thepresence of a related protein in vaginal fluid may representan additional line of defense against bacterial infection.

The presence of annexin-A1 and calgranulins A andB in human vaginal fluid during pregnancy suggests thatmechanisms exist to control immune cell influx into thecervical-vaginal compartment. Annexin A1 has anti-inflammatory properties that are mediated by its ability tointerfere with neutrophil migration.49 Calgranulin A and B,on the other hand, form a heterodimeric complex(calprotectin) that has both antimicrobial and inflamma-tory cell chemotactic activities.50 Elevated amniotic fluidcalprotectin levels are reported to be associated withintra-amniotic inflammation,48 although more recentevidence has questioned this relationship.10 SELDI-basedproteomics have also suggested that elevated calgranulinlevels are associated with intra-amniotic infection; calgran-ulins A and B were shown to be significantly elevated inamniotic fluid from women with intra-amniotic infection,9

and calgranulin B was detected only in amniotic fluid fromhumans and nonhuman primates with intra-amnioticinfection.6,7 Our data demonstrate that both calgranulin Aand B are present in vaginal fluid from 6 of 7 patients whopresented with premature labor without obvious evidenceof genital tract infection or inflammation.Thus, productionof calgranulin B, and possibly the calprotectin complex, in

vaginal fluid may be regulated differently from that ofamniotic fluid. The basis of this differential regulation isunknown, but calgranulins are produced by immune cellsincluding neutrophils, monocytes, and macrophages as wellas by epithelial and endothelial cells.51 Thus, it possible thatactivated immune cells are the source of calgranulins inamniotic fluid from patients with intra-amniotic infection,whereas epithelial cells of the urogenital tract are likely tobe the source of these proteins in vaginal fluid.

CONCLUSION

We have developed a simple and reliable mass spectrometry–based profiling approach to define the human vaginalfluid proteome and have used it to obtain an initial pro-file of vaginal fluid proteins in patients with pretermlabor.Ascending vaginal tract infections are known to becritical risk factors for preterm birth.52 Consequently,knowledge of vaginal fluid proteomic profiles of womenwith and without vaginal fluid infections is likely to pro-vide insight into the molecular processes associated withprematurity. In addition, because vaginal fluid has a rela-tively simple composition and can be sampled by mini-mally invasive procedures that do not pose significantrisks to mother or fetus, it lends itself to the possibilityof using proteomics to identify global longitudinalchanges in individual patient samples, thereby improvingchances of defining the pathological processes that leadto premature birth or other adverse outcomes and ulti-mately identifying biomarkers of patients at risk ofdeveloping such complications during pregnancy.

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Shotgun proteomic analysis of vaginal fluid from women in late pregnancy

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