Publications by authors named "Samir V Deshpande"

10 Publications

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Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Genes (Basel) 2019 07 30;10(8). Epub 2019 Jul 30.

US Army, CCDC-Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA.

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.
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http://dx.doi.org/10.3390/genes10080578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723491PMC
July 2019

Extracellular protein biomarkers for the characterization of enterohemorrhagic and enteroaggregative Escherichia coli strains.

J Microbiol Methods 2014 Mar 31;98:76-83. Epub 2013 Dec 31.

Research and Technology Directorate, Edgewood Chemical Biological Center, US Army Aberdeen Proving Ground, MD 21010, United States. Electronic address:

The extracellular proteins (ECPs) of enterohemorrhagic Escherichia coli (EHEC) can cause hemorrhagic colitis which may cause life threatening hemolytic-uremic syndrome, while that of enteroaggregative E. coli (EAEC) can clump to intestinal membranes. Liquid chromatography-electrospray ionization-tandem mass spectrometry based proteomics is used to evaluate a preliminary study on the extracellular and whole cell protein extracts associated with E. coli strain pathogenicity. Proteomics analysis, which is independent of genomic sequencing, of EAEC O104:H4 (unsequenced genome) identified a number of proteins. Proteomics of EHEC O104:H4, causative agent of the Germany outbreak, showed a closest match with E. coli E55989, in agreement with genomic studies. Dendrogram analysis separated EHEC O157:H7 and EHEC/EAEC O104:H4. ECP analysis compared to that of whole cell processing entails few steps and convenient experimental extraction procedures. Bacterial characterization results are promising in exploring the impact of environmental conditions on E. coli ECP biomarkers with a few relatively straightforward protein extraction steps.
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http://dx.doi.org/10.1016/j.mimet.2013.12.017DOI Listing
March 2014

A protein processing filter method for bacterial identification by mass spectrometry-based proteomics.

J Proteome Res 2011 Feb 20;10(2):907-12. Epub 2010 Dec 20.

SAIC, Aberdeen Proving Ground, Maryland 21010, USA.

A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.
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http://dx.doi.org/10.1021/pr101086aDOI Listing
February 2011

Iridovirus and microsporidian linked to honey bee colony decline.

PLoS One 2010 Oct 6;5(10):e13181. Epub 2010 Oct 6.

Division of Biological Sciences, The University of Montana, Missoula, Montana, United States of America.

Background: In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.

Methodology/principal Findings: We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006-2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.

Conclusions/significance: These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013181PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950847PMC
October 2010

Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics.

J Proteome Res 2010 Jul;9(7):3647-55

SAIC, Aberdeen Proving Ground, Maryland 21010, US. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010-5424, USA.

Whole cell protein and outer membrane protein (OMP) extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest neighbor database organism strains. These extracts were isolated from pathogenic and nonpathogenic strains of Yersinia pestis and Escherichia coli using ultracentrifugation and a sarkosyl extraction method followed by protein digestion and analysis using liquid chromatography tandem mass spectrometry (MS). Whole cell protein extracts contain many different types of proteins resident in an organism at a given phase in its growth cycle. OMPs, however, are often associated with virulence in Gram-negative pathogens and could prove to be model biomarkers for strain differentiation among bacteria. The mass spectra of bacterial peptides were searched, using the SEQUEST algorithm, against a constructed proteome database of microorganisms in order to determine the identity and number of unique peptides for each bacterial sample. Data analysis was performed with the in-house BACid software. It calculated the probabilities that a peptide sequence assignment to a product ion mass spectrum was correct and used accepted spectrum-to-sequence matches to generate a sequence-to-bacterium (STB) binary matrix of assignments. Validated peptide sequences, either present or absent in various strains (STB matrices), were visualized as assignment bitmaps and analyzed by the BACid module that used phylogenetic relationships among bacterial species as part of a decision tree process. The bacterial classification and identification algorithm used assignments of organisms to taxonomic groups (phylogenetic classification) based on an organized scheme that begins at the phylum level and follows through the class, order, family, genus, and species to the strain level. For both Gram-negative organisms, the number of unique distinguishing proteins arrived at by the whole cell method was less than that of the OMP method. However, the degree of differentiation measured in linkage distance units on a dendrogram with the OMP extract showed similar or significantly better separation than the whole cell protein extract method between the sample and correct database match compared to the next nearest neighbor. The nonpathogenic Y. pestis A1122 strain used does not have its genome available, and thus, data analysis resulted in an equal similarity index to the nonpathogenic 91001 and pathogenic Antiqua and Nepal 516 strains for both extraction methods. Pathogenic and nonpathogenic strains of E. coli were correctly identified with both protein extraction methods, and the pathogenic Y. pestis CO92 strain was correctly identified with the OMP procedure. Overall, proteomic MS proved useful in the analysis of unique protein assignments for strain differentiation of E. coli and Y. pestis. The power of bacterial protein capture by the whole cell protein and OMP extraction methods was highlighted by the data analysis techniques and revealed differentiation and similarities between the two protein extraction approaches for bacterial delineation capability.
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http://dx.doi.org/10.1021/pr100402yDOI Listing
July 2010

Double-blind characterization of non-genome-sequenced bacteria by mass spectrometry-based proteomics.

Appl Environ Microbiol 2010 Jun 2;76(11):3637-44. Epub 2010 Apr 2.

Edgewood Area, Gunpowder Branch, Science Applications International Corporation, Aberdeen Proving Ground, MD 21010-5424, USA.

Due to the possibility of a biothreat attack on civilian or military installations, a need exists for technologies that can detect and accurately identify pathogens in a near-real-time approach. One technology potentially capable of meeting these needs is a high-throughput mass spectrometry (MS)-based proteomic approach. This approach utilizes the knowledge of amino acid sequences of peptides derived from the proteolysis of proteins as a basis for reliable bacterial identification. To evaluate this approach, the tryptic digest peptides generated from double-blind biological samples containing either a single bacterium or a mixture of bacteria were analyzed using liquid chromatography-tandem mass spectrometry. Bioinformatic tools that provide bacterial classification were used to evaluate the proteomic approach. Results showed that bacteria in all of the double-blind samples were accurately identified with no false-positive assignment. The MS proteomic approach showed strain-level discrimination for the various bacteria employed. The approach also characterized double-blind bacterial samples to the respective genus, species, and strain levels when the experimental organism was not in the database due to its genome not having been sequenced. One experimental sample did not have its genome sequenced, and the peptide experimental record was added to the virtual bacterial proteome database. A replicate analysis identified the sample to the peptide experimental record stored in the database. The MS proteomic approach proved capable of identifying and classifying organisms within a microbial mixture.
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http://dx.doi.org/10.1128/AEM.00055-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876470PMC
June 2010

Discrimination and phylogenomic classification of Bacillus anthracis-cereus-thuringiensis strains based on LC-MS/MS analysis of whole cell protein digests.

Anal Chem 2010 Jan;82(1):145-55

Science Applications International Corporation, Aberdeen Proving Ground, Maryland 21010-0068, USA.

Modern taxonomy, diagnostics, and forensics of bacteria benefit from technologies that provide data for genome-based classification and identification of strains; however, full genome sequencing is still costly, lengthy, and labor intensive. Therefore, other methods are needed to estimate genomic relatedness among strains in an economical and timely manner. Although DNA-DNA hybridization and techniques based on genome fingerprinting or sequencing selected genes like 16S rDNA, gyrB, or rpoB are frequently used as phylogenetic markers, analyses of complete genome sequences showed that global measures of genome relatedness, such as the average genome conservation of shared genes, can provide better strain resolution and give phylogenies congruent with relatedness revealed by traditional phylogenetic markers. Bacterial genomes are characterized by a high gene density; therefore, we investigated the integration of mass spectrometry-based proteomic techniques with statistical methods for phylogenomic classification of bacterial strains. For this purpose, we used a set of well characterized Bacillus cereus group strains isolated from poisoned food to describe a method that relies on liquid chromatography-electrospray ionization-tandem mass spectrometry of tryptic peptides derived from whole cell digests. Peptides were identified and matched to a prototype database (DB) of reference bacteria with fully sequenced genomes to obtain their phylogenetic profiles. These profiles were processed for predicting genomic similarities with DB bacteria estimated by fractions of shared peptides (FSPs). FSPs served as descriptors for each food isolate and were jointly analyzed using hierarchical cluster analysis methods for revealing relatedness among investigated strains. The results showed that phylogenomic classification of tested food isolates was in consonance with results from established genomic methods, thus validating our findings. In conclusion, the proposed approach could be used as an alternative method for predicting relatedness among microbial genomes of B. cereus group members and potentially may circumvent the need for whole genome sequencing for phylogenomic typing of strains.
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http://dx.doi.org/10.1021/ac9015648DOI Listing
January 2010

Mass Spectrometry and Integrated Virus Detection System Characterization of MS2 Bacteriophage.

Toxicol Mech Methods 2007 ;17(5):241-54

Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA.

ABSTRACT In this study, we demonstrate the effect of sample matrix composition of MS2 virus on its characterization by ESI-MS and IVDS. MS2 samples grown and purified using various techniques showed different responses on ESI-MS than that on IVDS. The LC-MS of the specific biomarker of MS2 bacteriophage from an infected Escherichia coli sample was characterized by the presence of E. coli proteins. The significant impact of sample matrix was observed upon identification of MS2 using a database search. Infected E. coli with MS2 showed a matching score indifferent from uninfected ones. Only purified MS2, using CsCl and analyzed by LS-MS, showed a positive match using the database search. However, the variation in MS2 sample matrix had no effect on the deification of MS2.
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http://dx.doi.org/10.1080/15376510601123195DOI Listing
October 2012

Mass spectrometry-based proteomics combined with bioinformatic tools for bacterial classification.

J Proteome Res 2006 Jan;5(1):76-87

Geo-Centers, Inc., Aberdeen Proving Ground, Maryland 21010-0068, USA.

Timely classification and identification of bacteria is of vital importance in many areas of public health. We present a mass spectrometry (MS)-based proteomics approach for bacterial classification. In this method, a bacterial proteome database is derived from all potential protein coding open reading frames (ORFs) found in 170 fully sequenced bacterial genomes. Amino acid sequences of tryptic peptides obtained by LC-ESI MS/MS analysis of the digest of bacterial cell extracts are assigned to individual bacterial proteomes in the database. Phylogenetic profiles of these peptides are used to create a matrix of sequence-to-bacterium assignments. These matrixes, viewed as specific assignment bitmaps, are analyzed using statistical tools to reveal the relatedness between a test bacterial sample and the microorganism database. It is shown that, if a sufficient amount of sequence information is obtained from the MS/MS experiments, a bacterial sample can be classified to a strain level by using this proteomics method, leading to its positive identification.
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http://dx.doi.org/10.1021/pr050294tDOI Listing
January 2006

Detecting Bacteria by Direct Counting of Structural Protein Units by IVDS and Mass Spectrometry.

Toxicol Mech Methods 2006 ;16(9):485-93

Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA.

This report explores the direct counting of "hair-like" struc-tures specific for Gram-positive bacteria. Indications show that these structures are intact after removal from the cell and are sufficiently different from species to species of bacteria to give an indication of bacteria type if not actual identification. Their detection would represent a new approach to bacteria detection and identification. This report documents the detection of the bacterial structures using the physical nanometer counting methodology in the Integrated Virus Detection System (IVDS) and electrospray ionization-mass spectrometry.
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http://dx.doi.org/10.1080/15376510600910477DOI Listing
October 2012