Mass spectrometry peptides

Short peptide sequences predicted from mass spectrometry analysis

Displaying 1 - 8 of 8
Organisms: 

Description of data

Annotation of these sequenced genomes is a complex task, especially in case of eukaryotic genomes. Along with the use of prediction programs, manual curation is required to achieve accurate gene annotation. Although combination of prediction strategies are used for genome annotation, prediction of small genes, intron-exon boundaries and alternative transcripts remain challenging. We present high-resolution mass spectrometry based proteomics as a complementary approach for refining genome annotation. In this 'proteogenomic' analysis, peptide sequences obtained from mass spectrometry based shot-gun sequencing are mapped back to the genome and used for gene prediction in the way similar to using cDNA/ EST sequences.

For the mass spectrometry based analysis of proteome, proteins are digested into peptides and the sequence is deduced from fragment ion spectra derived from individual peptides. We carried out a comprehensive mass spectrometry analysis of proteins isolated from larvae, pupae and various adult mosquito tissues namely, midgut, salivary gland, ovary, malpighian tubules, testis, male accessory organs, head and viscera. All the analyses were carried out on Fourier transform mass spectrometer using high resolution MS and MS/MS parameter settings. We analyzed the mass spectrometry derived data using Mascot search algorithm with data deconvolution against protein database and six frame translation of genome of Anopheles gambiae. Peptides which were mapped to part of the genome where no CDS is annotated were categorized under different region such as. intergenic region, intron and UTR, were used to refine the gene annotation. Novel splice isoforms were identified using exon junction peptide database for hypothetical alternative transcripts. This large scale proteogenomics analysis led to the identification of many novel genes, novel splice isoforms as well as corrected and validated gene annotations in Anopheles gambiae genome.

Reference

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry.
Chaerkady R, Kelkar DS, Muthusamy B, Kandasamy K, Dwivedi SB, Sahasrabuddhe NA, Kim MS, Renuse S, Pinto SM, Sharma R, Pawar H, Sekhar NR, Mohanty AK, Getnet D, Yang Y, Zhong J, Dash AP, MacCallum RM, Delanghe B, Mlambo G, Kumar A, Prasad TS, Okulate M, Kumar N, Pandey A.
Genome Res. 2011 Sep 30.

Whole head, compound eyes, total head appendages, and antennae proteomes of the Anopheles gambiae mosquito

Description of data

This study explored the protein composition of specific Anopheles gambiae mosquito head tissue isolates, with particular relevance to brain function, and the sensory modalities of vision, olfaction and gustation. We characterized the proteomes of isolated adult female An. gambiae mosquito tissues using a combination LC/MS/MS and targeted LC/MRM analysis; specifically whole mosquito head with all appendages attached, compound eyes, total head appendages (THAs; maxillary palps, antennae and proboscises), and antennae. Proteomics data were acquired on single-dimension LC/MS/MS runs using an LTQ Velos Orbitrap instrument (Thermo Fisher Scientific). The combined injections from Anopheles tissue yielded 8230 peptide identifications at a 1% false discovery rate (FDR) and 8350 at a 5% FDR, and 1,322 non-redundant protein identifications. Many of these identifications were subsequently validated using targeted LC/MRM based approaches; making this one of the largest representative collections of isolated head tissue protein identifications in mosquitoes to-date, with tissue specific quantification and detection.

The data, including the search results are also deposited into the PeptideAtlas with accession PASS00300.

Accessing the data

Reference

Champion M.M., Sheppard A.D., Rund S.S.C., Freed S.A., O’Tousa J.E. and Duffield G.E. (2015) Qualitative and quantitative proteomics methods for the analysis of the Anopheles gambiae mosquito proteome. Chapter 2 in ‘Short views on Insect Genomics and Proteomics’ book (Editors: T. Adeniran, R. Chandrasekar and M. Goldsmith), within the ‘Entomology in Focus’ book series (Editor: F.L. Cônsoli) (Springer publishers).

Authors

Experimentalists: Matthew M. Champion, Aaron D. Sheppard, Samuel S.C. Rund, Stephanie A. Freed, Joseph E. O’Tousa and Giles E. Duffield, University of Notre Dame, Notre Dame, IN, USA
VectorBase data integration: Bob MacCallum

Organisms: 

Description of data

Olfaction is important for the mosquito detection of blood-feeding hosts, sugar feeding sources and oviposition sites.. In this study we characterized the proteome of adult female An. gambiae antennae, THAs (maxillary palps, antennae and proboscises) and whole mosquito head using LC/MS/MS analysis. MS and MS/MS data were acquired on an LTQ Velos Orbitrap instrument (Thermo Fisher Scientific) using a TOP8 method. The combined injections yielded 7201 peptide identifications at a 1% false discovery rate (FDR) and 8350 at a 5% FDR, representing one of the largest collections of sensory protein identifications in mosquitoes to-date.

The data is also deposited into the PeptideAtlas with accession PASS00300.

Access to the data

Reference

Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae.
Samuel SC Rund, Nicolle A. Bonar, Matthew M. Champion, John P. Ghazi, Cameron M. Houk, Matthew T. Leming, Zainulabeuddin Syed, and Giles E. Duffield
Sci Rep. 2013;3:2494.

Authors

Experimentalists: Matthew M. Champion, John P. Ghazi, Samuel S.C. Rund, and Giles E. Duffield, University of Notre Dame, Notre Dame, IN, USA
VectorBase data integration: Bob MacCallum

Organisms: 

Description of data

Anopheles gambiae has a well-adapted system for host localization, feeding, and mating behavior, which are all governed by neuronal processes in the brain. However, there are no published reports characterizing the brain proteome to elucidate neuronal signaling mechanisms in the vector. To this end, a large-scale mapping of the brain proteome of An. gambiae was carried out using high resolution tandem mass spectrometry, revealing a repertoire of >1800 proteins, of which 15% could not be assigned any function. A large proportion of the identified proteins were predicted to be involved in diverse biological processes including metabolism, transport, protein synthesis, and olfaction. This study also led to the identification of 10 GPCR classes of proteins, which could govern sensory pathways in mosquitoes. Proteins involved in metabolic and neural processes, chromatin modeling, and synaptic vesicle transport associated with neuronal transmission were predominantly expressed in the brain. Proteogenomic analysis expanded our findings with the identification of 15 novel genes and 71 cases of gene refinements, a subset of which were validated by RT-PCR and sequencing.

Reference

Brain proteomics of Anopheles gambiae.
Dwivedi SB, Muthusamy B, Kumar P, Kim MS, Nirujogi RS, Getnet D, Ahiakonu P, De G, Nair B, Gowda H, Prasad TS, Kumar N, Pandey A, Okulate M.
OMICS. 2014 Jul;18(7):421-37

Organisms: 

Description of data

High resolution LC-MS/MS was used to analyze the proteome of Ixodes scapularis (Lyme disease tick) embryonic ISE6 cells following infection with Langat virus (LGTV) and identify proteins associated with viral infection and replication. Maximal LGTV infection of cells and determination of peak release of infectious virus, was observed at 36 hours post infection (hpi). Proteins were extracted from ISE6 cells treated with LGTV and non-infectious (UV inactivated) LGTV at 36 hpi and analyzed by mass spectrometry. The Omics Discovery Pipeline (ODP) identified thousands of MS peaks. Protein homology searches against the I. scapularis IscaW1 genome assembly identified a total of 486 proteins that were subsequently assigned to putative functional pathways using searches against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. 266 proteins were differentially expressed following LGTV infection relative to non-infected (mock) cells. Of these, 68 proteins exhibited increased expression and 198 proteins had decreased expression. The majority of the former were classified in the KEGG pathways: "translation", "amino acid metabolism", and "protein folding/sorting/degradation". Finally, Trichostatin A and Oligomycin A increased and decreased LGTV replication in vitro in ISE6 cells, respectively.

Reference

Changes in the Proteome of Langat-Infected Ixodes scapularis ISE6 Cells: Metabolic Pathways Associated with Flavivirus Infection.
Grabowski JM, Perera R, Roumani AM, Hedrick VE, Inerowicz HD, Hill CA, Kuhn RJ
PLoS Negl Trop Dis. 2016 Feb 9;10(2):e0004180.

Organisms: 

Description of data

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.

Reference

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry.
He N, Botelho JM, McNall RJ, Belozerov V, Dunn WA, Mize T, Orlando R, Willis JH.
Insect Biochem Mol Biol. 2007 Feb;37(2):135-46.

Organisms: 

Description of data

A proteomic analysis of salivary glands from female An. gambiae mosquitoes. Salivary gland extracts were digested with trypsin using two complementary approaches and analyzed by LC-MS/MS. This led to identification of 69 unique proteins, 57 of which were novel. We carried out a functional annotation of all proteins identified in this study through a detailed bioinformatics analysis. Even though a number of cDNA and Edman degradation-based approaches to catalog transcripts and proteins from salivary glands of mosquitoes have been published previously, this is the first report describing the application of MS for characterization of the salivary gland proteome.

Reference

A proteomic analysis of salivary glands of female Anopheles gambiae mosquito.
Kalume DE, Okulate M, Zhong J, Reddy R, Suresh S, Deshpande N, Kumar N, Pandey A.
Proteomics. 2005 Sep;5(14):3765-77.

Organisms: 

Description of data

3,967 mass spectra from 16 LC-MS/MS runs of An. gambiae salivary gland homogenates against the An. gambiae genome database. This allowed us to validate 23 known transcripts and 50 novel transcripts. In addition, a novel gene was identified on the basis of peptides that matched a genomic region where no gene was known and no transcript had been predicted. The amino termini of proteins encoded by two predicted transcripts were confirmed based on N-terminally acetylated peptides sequenced by tandem mass spectrometry. Finally, six sequence polymorphisms could be annotated based on experimentally obtained peptide sequences.

Reference

Genome annotation of Anopheles gambiae using mass spectrometry-derived data.
Kalume DE, Peri S, Reddy R, Zhong J, Okulate M, Kumar N, Pandey A.
BMC Genomics. 2005 Sep 19;6:128.

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