Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPsEf and Stx-SSPsEf). Five selected toxic dinoflagellates, Alexandrium minutum, A. lusitanicum, A. tamarense, Gymnodinium catenatum, and Karenia mikimotoi, were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPsEf and Stx-SSPsEf in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

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Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

10.1101/2021.07.28.454135 ◽

2021 ◽

Author(s):

Anna Cioce ◽

Beatriz Calle ◽

Andrea Marchesi ◽

Ganka Bineva-Todd ◽

Helen Flynn ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Surface ◽

Cell Line ◽

Specific Protein ◽

Surface Proteins ◽

Cancer Development ◽

Biological Processes ◽

Culture Studies ◽

Cell Surface Proteins ◽

A Cell

Interactions between cells fundamentally impact biological processes. In cancer development, such interactions define key stages of disease that cannot be adequately recapitulated in cell monoculture. Complex co-culture studies have been key to unraveling the complexity of these processes, usually by sorting cells and transcriptome or bulk proteome analyses. However, these methods invariably lead to sample loss and do not capture aberrant glycosylation as an important corollary of cancer formation. Here, we report the development of Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped with a biosynthetic AND gate that uses bioorthogonally tagged sugars to generate glycosylation precursors. The cellular glycosylation machinery then introduces bioorthogonal tags into glycoproteins exclusively in cell lines expressing the enzymes of the biosynthetic AND gate. Modification with clickable reporter moieties allows for imaging or enrichment with mass spectrometry-proteomics in a cell-specific fashion. Making use of glycans as a property of most cell surface proteins, we use BOCTAG as an efficient means for cell-specific protein tracing.

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Implementation of MALDI-TOF Mass Spectrometry and Peak Analysis: Application to the Discrimination of Cryptococcus Species and Their Interspecies Hybrids

10.20944/preprints202007.0423.v1 ◽

2020 ◽

Author(s):

Margarita E. Zvezdanova ◽

Manuel J. Arroyo ◽

Gema Méndez ◽

Jesús Guinea ◽

Luis Mancera ◽

...

Keyword(s):

Mass Spectrometry ◽

Ad Hoc ◽

Specific Protein ◽

Rapid Identification ◽

Its2 Region ◽

Bacterial Typing ◽

Maldi Tof ◽

Interspecies Hybrids ◽

Species Specific ◽

Correct Discrimination

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of Flight) is a type of mass spectrometry (MS) that has been widely implemented for the rapid identification of microorganisms over the last decade. The accuracy and flexibility of this method has encouraged researchers to implement the analysis of protein spectra obtained by MALDI-TOF for the discrimination of close-related species and bacterial typing. In this study, a standardized methodology based on the detection of species-specific protein peaks from the spectra obtained with MALDI-TOF is described. The methodology was applied to a collection of Cryptococcus spp. (n=70) previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region. An expanded ad-hoc database was also built for their discrimination with MALDI-TOF. This approach did not allow the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of the PLS-DA and SVM algorithms in a two-step analysis allowed 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. Besides, PCA analysis prior to SVM provided 98.45% correct discrimination of the 3 analyzed species in a one-step analysis. The method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.

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PhyloQuant approach provides insights into Trypanosoma cruzi evolution using a systems-wide mass spectrometry-based quantitative protein profile

Communications Biology ◽

10.1038/s42003-021-01762-6 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Simon Ngao Mule ◽

André Guilherme Costa-Martins ◽

Livia Rosa-Fernandes ◽

Gilberto Santos de Oliveira ◽

Carla Monadeli F. Rodrigues ◽

...

Keyword(s):

Mass Spectrometry ◽

Trypanosoma Cruzi ◽

Large Scale ◽

Clinical Course ◽

Expression Profiles ◽

Protein Profile ◽

Ecological Distribution ◽

Specific Proteins ◽

Species Specific ◽

Character Mapping

AbstractThe etiological agent of Chagas disease, Trypanosoma cruzi, is a complex of seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution requires understanding the parasite origin and population structure. In this study, we introduce the PhyloQuant approach to infer the evolutionary relationships between organisms based on differential mass spectrometry-based quantitative features. In particular, large scale quantitative bottom-up proteomics features (MS1, iBAQ and LFQ) were analyzed using maximum parsimony, showing a correlation between T. cruzi DTUs and closely related trypanosomes’ protein expression and sequence-based clustering. Character mapping enabled the identification of synapomorphies, herein the proteins and their respective expression profiles that differentiate T. cruzi DTUs and trypanosome species. The distance matrices based on phylogenetics and PhyloQuant clustering showed statistically significant correlation highlighting the complementarity between the two strategies. Moreover, PhyloQuant allows the identification of differentially regulated and strain/DTU/species-specific proteins, and has potential application in the identification of specific biomarkers and candidate therapeutic targets.

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Development of a C12Im-Cl-assisted method for rapid sample preparation in proteomic application

Analytical Methods ◽

10.1039/d0ay02079f ◽

2021 ◽

Author(s):

Chang Liu ◽

Xiaoxia Si ◽

Shumei Yan ◽

Xinyuan Zhao ◽

Xiaohong Qian ◽

...

Keyword(s):

Mass Spectrometry ◽

Sample Preparation ◽

Protein Extraction ◽

Sample Processing ◽

Processing Methods ◽

Proteomic Analyses

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods, including protein extraction and digestion, before MS remain as bottlenecks in the...

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SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates

Bioinformatics ◽

10.1093/bioinformatics/btaa092 ◽

2020 ◽

Vol 36 (11) ◽

pp. 3447-3456 ◽

Cited By ~ 2

Author(s):

Matthew Waas ◽

Shana T Snarrenberg ◽

Jack Littrell ◽

Rachel A Jones Lipinski ◽

Polly A Hansen ◽

...

Keyword(s):

Cell Surface ◽

Specific Surface ◽

Web Application ◽

Rank Order ◽

Surface Proteins ◽

Supplementary Information ◽

Live Cells ◽

Specific Marker ◽

Cell Type ◽

Cell Type Specific

Abstract Motivation Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing ‘omic’ discovery datasets is the selection of candidate markers that are most applicable for downstream applications. Results Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. Availability and implementation Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

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Magnetic Bead-Based Salivary Peptidome Profiling Analysis for Severe Early Childhood Caries

Caries Research ◽

10.1159/000360868 ◽

2014 ◽

Vol 49 (1) ◽

pp. 63-69 ◽

Cited By ~ 8

Author(s):

Yan Si ◽

Shuang Ao ◽

Weijian Wang ◽

Feng Chen ◽

Shuguo Zheng

Keyword(s):

Mass Spectrometry ◽

Early Childhood ◽

Early Childhood Caries ◽

Expression Profiles ◽

Salivary Protein ◽

Preschool Age ◽

Diagnostic Model ◽

Peptide Mass ◽

Severe Early Childhood Caries ◽

Childhood Caries

Objective: To investigate the differential salivary protein expression profiles between children with severe early childhood caries (S-ECC) and caries-free (CF) children at the age of 3 years. Methods: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with weak cation exchange magnetic beads, and peptide mass fingerprints were created by scanning mass spectrometry signals. Salivary samples from 20 children were analyzed (10 for each group). Results: Eleven protein peaks were significantly different (p < 0.05) between the two groups. Eight of these peaks were higher in the S-ECC group and three were higher in the CF group. To establish a diagnostic model for discrimination between the two groups, we chose three peptides (3,186.2, 3,195.8 and 3,324.8 Da) that exhibited the best fitted curve, by which the two groups were better separated when compared with other combinations. Conclusions: The salivary biomarkers identified revealed significant differences between the CF and the S-ECC group. Our results provide novel insight into the salivary protein profile of preschool-age children with dental caries and may lead to the development of a new strategy for screening high-risk populations.

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Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽

10.1098/rsob.110010 ◽

2011 ◽

Vol 1 (3) ◽

pp. 110010 ◽

Cited By ~ 51

Author(s):

Clive Metcalfe ◽

Peter Cresswell ◽

Laura Ciaccia ◽

Benjamin Thomas ◽

A. Neil Barclay

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Cell Surface ◽

Disulfide Bonds ◽

Reduction Process ◽

Cysteine Residue ◽

Surface Proteins ◽

Protein Activity ◽

Functional Changes ◽

Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

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Fluoride exposure inhibits protein expression and enzyme activity in the lung-stage larvae ofAscaris suum

Parasitology ◽

10.1017/s0031182006000576 ◽

2006 ◽

Vol 133 (4) ◽

pp. 497-508 ◽

Cited By ~ 3

Author(s):

M. K. ISLAM ◽

T. MIYOSHI ◽

M. YAMADA ◽

M. A. ALIM ◽

X. HUANG ◽

...

Keyword(s):

Protein Expression ◽

Expression Profiles ◽

Immunoblot Analysis ◽

Specific Protein ◽

Inorganic Pyrophosphatase ◽

2D Electrophoresis ◽

Peptide Mass ◽

Enzyme Families ◽

Protein Expression Profiles ◽

Peptide Mass Spectrometry

Sodium fluoride (NaF) is an anion that has been previously shown to block the moulting process ofAscaris suumlarvae. This study describes moulting and development-specific protein expression profiles ofA. suumlung-stage L3 (AsLL3) following NaF exposure. AsLL3s cultured in the presence or absence of NaF were prepared for protein analysis using two-dimensional (2D) electrophoresis. NaF exposure inhibited at least 22 proteins in AsLL3 compared with moulted larvae (i.e. AsLL4). A further comparison of AsLL4 with those of pre-cultured AsLL3 and NaF-exposed AsLL3 revealed 8 stage-specifically and 4 over-expressed proteins. Immunoblot analysis revealed an inhibition by NaF of 19 immunoreactive proteins. Enzyme assay and immunochemical data showed an inhibition of the moulting-specific inorganic pyrophosphatase activity by 41% and a decreased expression in NaF-treated larvae, indicating its significance in the moulting process. A protein spot associated with NaF inhibition was isolated and identified by peptide mass spectrometry and bioinformatics approaches to be a member of 3–hydroxyacyl–CoA dehydrogenase/short-chain dehydrogenase enzyme families. These results have implications for the identification of proteins specific to the moulting process as potential chemotherapeutic targets.

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Preparation and characterisation of an 57Fe enriched haemoglobin spike material for species-specific isotope dilution mass spectrometry

Journal of Analytical Atomic Spectrometry ◽

10.1039/c6ja00028b ◽

2016 ◽

Vol 31 (9) ◽

pp. 1846-1857 ◽

Cited By ~ 6

Author(s):

C. Brauckmann ◽

C. Frank ◽

D. Schulze ◽

P. Kaiser ◽

R. Stosch ◽

...

Keyword(s):

Mass Spectrometry ◽

Isotope Dilution ◽

Isotope Dilution Mass Spectrometry ◽

Icp Ms ◽

Species Specific

A species-specific ID-ICP-MS method for intact haemoglobin was developed applying an 57Fe enriched haemoglobin spike, which was prepared and characterised carefully.

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The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

Biochemical Journal ◽

10.1042/bj3220049 ◽

1997 ◽

Vol 322 (1) ◽

pp. 49-56 ◽

Cited By ~ 36

Author(s):

Philemon PAPANASTASIOU ◽

Malcolm J. McCONVILLE ◽

Julie RALTON ◽

Peter KÖHLER

Keyword(s):

Specific Surface ◽

Acyl Chain ◽

Giardia Duodenalis ◽

Compositional Analysis ◽

Surface Protein ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Surface Proteins ◽

Phase Partitioning ◽

Chromatography Analysis

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

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