Metaproteomic analysis of human cervical-vaginal fluid in residual Pap tests:

Insights into the cervical microbiome

1Somi Afiuni-Zadeh; 2Pratik Jagtap; 3Timothy J. Griffin; 4Marnie L. Peterson; 1Amy P.N. Skubitz 1Department of Lab Medicine and Pathology; 2Center for Mass Spectrometry and Proteomics; 3Department of Biochemistry, Molecular Biology and Biophysics,

4Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN

The complex proteome of human cervical-vaginal fluid contains human proteins as well as proteins from microbial communities that colonize the cervical-vaginal area. These microorganisms may prevent or predispose women to gynecological conditions and malignancies. The liquid Pap test fixative is a novel and ideal source for the discovery of human and microbial biomarkers since it is readily available, collected near the site of origin, and may not contain the high abundance proteins found in blood that could mask potential biomarkers1. In this study, we developed a novel protein database search method encompassing microbes likely present in cervical-vaginal fluid, and used the Minnesota Supercomputing Institute (MSI) Galaxy-P platform to characterize microbial proteins in residual Pap test fixatives from 4 individual women with normal cytology as well as a pool of 40 normal samples using mass spectrometry techniques.

BACKGROUND

Acetone precipitation of

proteins

Filter Aided Sample Preparation( FASP)

Trypsin

Peptide mixture

Size and quantity of proteins determined by 1-D SDS-PAGE and silver staining of gels

HPLC high pH fractionation

SurePathTM Preservative Fluid: 21.7% ethanol, 1.2% methanol, 1.1% isopropanol

METHODS

Protein mixture

Routine Pap test Protein concentration determined by BCA

LTQ Orbitrap Velos Mass Spectrometry

1. Boylan KLM, Afiuni-Zadeh S, Geller MA, Hickey K, Griffin TJ, Pambuccian SE, Skubitz APN, (2014) Clinical Proteomics 11 (30)

2. Shilov IV, Seymour SL, Patel AA, Loboda A, Tang WH, Keating SP, Hunter CL, Nuwaysir LM, Schaeffer DA, (2007) Molecular & Cellular Proteomics 6:9, 1638-1655.

3. Mesuere et al. (2015) Proteomics doi:10.1002/pmic.201400361 2015 Universiteit Gent

4. Huson D. et al 17:377–386 ©2007 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/07; www.genome.org

5. P Jagtap, J Goslinga, JA Kooren, T McGowan, MS Wroblewski, et al. Proteomics 13 (8), 1352-1357

REFERENCES

CONCLUSIONS

RESULTS (MEGAN Analysis) RESULTS (Unipept Analysis)

Phylum level assignments of normal Pap metaproteome (n=346)

Genus level assignments of normal Pap metaproteome (n=295)

Genus Taxonomy Profile

Lactobacillus

Actinomyces

Candida<Debaryomycetaceae>Fusobacterium

Acinetobacter

Gardnerella

Corynebacterium

Sphingobacterium

Mycobacterium

Treponema

Staphylococcus

Veillonella

Mobiluncus

Propionibacterium

Anaerococcus

Escherichia

Porphyromonas

Peptoniphilus

Roseomonas

SEED Functions of normal Pap metaproteome (n=182)

SEED Function Profile Carbohydrates

Stress Response

Cofactors, Vitamins, Prosthetic Groups,Pigments

Protein Metabolism

Amino Acids and Derivatives

Motility and Chemotaxis

Clustering-based subsystems

Phages, Prophages, Transposableelements, Plasmids

Cell Wall and Capsule

Phosphorus Metabolism

Respiration

Nucleosides and Nucleotides

Membrane Transport

Virulence, Disease and Defense

Nitrogen Metabolism

DNA Metabolism

RNA Metabolism

Sulfur Metabolism

Fatty Acids, Lipids, and Isoprenoids

Funding for this project was provided by the Minnesota Ovarian Cancer Alliance, CanCurables Foundation, and a UM Masonic Cancer Center Brainstorm Award. UM Core Facilities: The Center for Mass Spectrometry and Proteomics, the Minnesota Cancer Center, the Minnesota Supercomputing Institute, and the BioNet Tissue Procurement Facility (supported by NIH grant P30-CA77598 and the Academic Health Center). Galaxy-P was supported by NSF ABI Development Grant #1147079.

ACKNOWLEDGEMENTS

KEGG Functions of normal Pap metaproteome (n=243)

Peptide

Phylum

Genus

Species

AEDADDLSPSIVVSR

Actinobacteria

Hevea

Hevea brasiliensis

IVNEVDNVNR

Firmicutes

Lactobacillus

Lactobacillus crispatus

TAAGIVSLLGVK

Firmicutes

Anaerococcus

Anaerococcus lactolyticus

QAAAAPTQPAPK

Proteobacteria

Acinetobacter

KEGG Function Profile

Metabolism

Human Diseases

Genetic Information Processing

Unclassified

Environmental Information Processing

Organismal Systems

Cellular Processes

Using Unipept program, we have found 250 unique peptides in 4 individuals and pooled samples belonging to 7 phylum (Actinobacteria, Ascomycota, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes) similar to MEGAN analysis. Examples of peptides present in 3 of 4 individuals and their phylum, genus and species are shown below.

Search high accuracy MS/MS data against “target” version of human + human microbiome project (HMP) protein sequences and 3-frame cDNA protein sequences using ProteinPilot2 in Minnesota Super Computing Institute (MSI) GalaxyP platform

Accession numbers associated with all microbial peptides identifications from first search, were merged with human database plus contaminants to create a target decoy database so that false discovery rate (FDR) can be calculated in the second

step.

Refined target-decoy database of human + bacterial proteins and translated 3-frame cDNA sequences

Search high mass accuracy peak lists against refined database

Distinct peptide sequences from spectra identified at 5% local FDR

Validate high confidence peptides matches or potential alternative site isoforms via peptide spectra match (PSM) evaluator and analyzed with downstream metaprotomic analysis tools Unipept3 and MEGAN (KEGG and SEED)4 on BLAST searches of proteins

• We have successfully characterized the profile of microbial flora in phylum, family, genus and species level in Pap test of women with normal cytology. • Using MSI tools, we have developed a novel FASTA protein database consisting of translated reference genomes from 130 microbial organisms from Human Microbiome Project (HMP) concatenated with the human proteome database (UniProt isoform canonical database) and 3-frame cDNA protein sequences in human urogenital tract . • Using a two step search method4 for data analysis, avoids false positive matches, resulted in identification of higher number of high confidence peptides. • MEGAN5 (MetaGenomeAnalyzer) was used on BLAST searches of protein sequences to generate functional characterization of the metaproteome using SEED and KEGG pathway analysis. • Peptides corresponding to proteins involved in carbohydrate metabolism, stress response, cofactors, vitamins, protein metabolism, phosphorus metabolism, and respiration were assigned by the SEED functional profile. • Using the Unipept tool, we identified over 250 unique microbial peptides encompassing a wide variety of bacterial families, genera, and species including Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, and Spirochaetes. • Top 10 proteins in normal Pap metaproteome are mainly metabolism enzymes.

0 5 10 15 20 25 30

Glucose-6-phosphate isomerase

L-lactate dehydrogenase

D-lactate dehydrogenase

Ribosomal protein L11 methyltransferase

Beta-phosphoglucomutase

Phosphoglycerate kinase

Enolase

Fructose-bisphosphate aldolase class II

Acyl-CoA dehydrogenase

NAD-dependent glyceraldehyde-3-phosphate dehydrogenase

Number of hits

Top proteins in normal Pap metaproteome

Figure 1. MEGAN taxonomical analysis on normal Pap metaproteome consisting of 4 normal individuals plus the pool of 40 normal women; (A)phylum level assignment , (B)genus level assignment, (C)SEED functional profile , (D)KEGG functional profile ; n = number of hits; we found 283 hits assigned to 34 species.

A B

C D

Figure 2. Top proteins in normal Pap metaproteome assigned by SEED functional profile

Phylum Taxonomy Profile Firmicutes

Actinobacteria

Proteobacteria

Ascomycota

Fusobacteria

Bacteroidetes

Spirochaetes

63rd ASMS