Two‑Dimensional Proteome Profiling of Viral & Bacterial Strains

Two‑Dimensional Proteome Profiling of Viral & Bacterial Strains – High‑Resolution Characterisation & Differential Expression Mapping

You are searching for two‑dimensional protein profiling of strains because you need to perform this characterisation—whether to compare virulent vs. attenuated strain proteomes, identify strain‑specific protein markers, map post‑translational modifications (PTMs) in pathogen proteins, or understand host‑pathogen interactions at the proteome level. We provide a complete 2D proteome profiling service that combines high‑resolution 2D gel electrophoresis, DIGE (difference gel electrophoresis), and shotgun 2D‑LC‑MS/MS to deliver unambiguous protein identification, quantitative abundance ratios, and modification site localisation for any cultured strain.

What We Detect – Full Proteome Coverage, PTM Resolution & Differential Expression

Our two‑dimensional characterisation goes far beyond simple 1D SDS‑PAGE. Using immobilised pH gradient (IPG) strips (7‑24 cm, pH 3‑10, 4‑7, or 6‑11) for first‑dimension isoelectric focusing, followed by high‑porosity SDS‑PAGE (8‑20% gradient, 20×20 cm gels) for second‑dimension molecular weight separation, we resolve up to 5,000 protein spots per gel with pI resolution of 0.01 pH units and mass resolution of ±2 kDa. For comparative studies, we employ 2D‑DIGE (CyDye™ minimal labelling, Cy2/Cy3/Cy5) to co‑separate up to three samples on the same gel, eliminating inter‑gel variation and quantifying abundance differences down to 1.2‑fold (p<0.01, ANOVA). We also specialise in PTM‑targeted 2D methods: phosphoprotein staining (Pro‑Q Diamond) or phospho‑specific 2D (pre‑fractionation with TiO₂) for phosphorylation mapping; lectin blotting or periodic acid‑Schiff (PAS) staining for glycosylation profiling; and 2D‑Western blot with strain‑specific antibodies to confirm low‑abundance virulence factors. For membrane proteins, we apply 2D‑BN‑PAGE (blue native) combined with SDS‑PAGE to resolve protein complexes and their subunit composition.

How Deep We Go – From Spot Picking to Full MS Identification & Interaction Networks

We don't just produce gel images. Our advanced pipeline includes automated spot excision (Ettan™ Spot Picker, picking 500 spots/hour), in‑gel digestion (trypsin or Lys‑C, automated ProGest™), and nano‑LC‑MS/MS (Orbitrap Fusion™ Lumos, 120,000 resolution, HCD fragmentation) to identify each protein with ≥2 unique peptides, FDR <1%. For differential spots, we report fold change, peptide coverage (typically >25%), and PTM localization (via pFind or phosphoRS). We also perform 2D‑based comparative proteomics across multiple strains (e.g., wild‑type vs. mutant, virulent vs. attenuated, drug‑resistant vs. sensitive) using DeCyder™ or Progenesis SameSpots analysis – outputting principal component (PCA) and hierarchical clustering heatmaps of strain proteomes. Beyond identification, we offer protein‑protein interaction network mapping (STRING or IntAct) and pathway enrichment (KEGG, GO) to place differentially expressed proteins into functional context. For low‑abundance virulence factors, we perform pre‑fractionation (subcellular fractionation, membrane enrichment, or affinity pull‑down) before 2D separation, achieving detection of proteins present at <50 copies per cell.

Why Our 2D Strain Proteome Characterisation Stands Out – Biosafe Handling, Reproducibility & Deep Annotation

1. BSL‑2/BSL‑3 sample compatibility: We have dedicated BSL‑2 and BSL‑3 laboratories for safe inactivation and protein extraction from infectious viral (influenza, SARS‑CoV‑2, Ebola), bacterial (Mycobacterium tuberculosis, Brucella), and fungal strains. All inactivation protocols are validated to render samples non‑infectious before 2D analysis.
2. Strain‑optimised protein extraction: We use optimised lysis buffers (chaotropes + detergents + protease/phosphatase inhibitors) tailored to capsid/cell wall disruptors (sonication, bead beating, freeze‑thaw) to maximise yields and minimise degradation. Typical protein yields: 0.5‑2 mg from 10⁸ cells or 1 mL of allantoic fluid.
3. Ultra‑high reproducibility: Our automated 2D‑DIGE workflow achieves spot volume CV <15% across triplicate gels – essential for detecting genuine strain‑specific differences. We include internal pooled standard (in all Cy2 channel) for accurate normalisation.
4. Deep MS coverage & modification mapping: From a single 2D gel, we typically identify 400‑1,500 protein spots corresponding to 200‑600 unique proteins. For phosphorylation, we routinely localise sites down to single residue accuracy (Ascore ≥19). For glycoproteins, we map N‑glycosylation motifs (NXS/T) and O‑glycosites by MS/MS.
5. Reporting & data delivery: You receive high‑resolution gel images (TIF, 300 dpi), spot maps with numbered IDs, excel tables of quantified spot volumes, MS identification reports (protein name, accession, coverage, peptides, PTMs), statistical comparisons (t‑test/ANOVA), and functional network plots. All data are MIAPE (Minimum Information About a Proteomics Experiment) compliant.

Who Relies on Our 2D Strain Protein Profiling – Real‑World Impact

A vaccine development company used our 2D‑DIGE and MS to compare a live‑attenuated influenza strain with its wild‑type parent. We identified 12 haemagglutinin (HA) fragments and 8 neuraminidase (NA) variants with altered glycosylation – key to rational attenuation. Another client, studying Mycobacterium tuberculosis drug‑resistant clinical isolates, discovered a KatG mutation that was silent at the DNA level but caused a pI shift (from 5.9 to 6.4) on 2D gels, leading to catalase‑peroxidase inactivation and isoniazid resistance – missed by whole‑genome sequencing alone. A veterinary lab sent us Brucella abortus field strain vs. vaccine strain S19; our 2D phosphoproteome analysis revealed differential phosphorylation of two outer membrane proteins (Omp25 and Omp31) that correlated with attenuated virulence. An academic group studying SARS‑CoV‑2 variant proteomes used our 2D‑based host‑virus interaction mapping to show that the Delta variant induced stronger N‑glycosylation changes in the host ER‑Golgi network compared to the ancestral strain.

Ready to Run Your 2D Strain Protein Characterisation?

Send us live or inactivated strain material (≥10⁸ CFU for bacteria, ≥10⁷ PFU for viruses, or ≥1 mg total protein extract) – we accept pellets, culture supernatants, allantoic fluid, or infected cell lysates under appropriate biosafety packaging. We will perform protein extraction, 2D‑DIGE or 2D‑PAGE separation, spot detection and quantitation, automated spot picking, in‑gel digestion, LC‑MS/MS identification, PTM localisation, and full bioinformatic analysis – delivering a comprehensive report within 3‑5 weeks (depending on replicate number and gel count). Request a free consultation; we will design the optimal 2D workflow (global proteome, phosphoproteome, glycoproteome, or complexome) for your strain comparison or modification mapping goal.