Host–Pathogen Quantitative Proteomics Service

Q: How does quantitative mass spectrometry compare to RNA‑seq for host–pathogen infection studies?

While RNA‑seq measures transcriptional responses, it cannot evaluate post‑transcriptional host shutoff, viral‑induced ribosome degradation, or bacterial proteolysis of host defense proteins. Furthermore, RNA‑seq cannot quantify physical virulence toxins, bacterial secretion effectors, or post‑translational kinase signaling cascades (e.g., TBK1 or p38 phosphorylation). Quantitative mass spectrometry provides an unbiased readout of actual functional protein abundance and pathway rewiring in both the host and pathogen simultaneously.

Q: How do you prevent high‑abundance host proteins from masking low‑abundance pathogen proteins?

We utilize a combination of pre‑analytical and analytical strategies: (1) physical enrichment protocols, such as gentle differential centrifugation to pellet intact bacterial cells or organellar isolation of infected phagosomes; (2) high‑resolution 4D‑DIA mass spectrometry on the timsTOF Pro 2, where trapped ion mobility (TIMS) separates co‑eluting host and pathogen peptide ions across the collisional cross‑section (CCS) dimension; and (3) optimized microflow chromatography delivering exceptional peak capacity and high signal‑to‑noise ratios.

Q: What biosafety levels (BSL) can your facility accommodate for host–pathogen samples?

Creative Proteomics accepts samples derived from BSL‑1 and BSL‑2 biological agents. For pathogens classified under BSL‑3 (such as SARS‑CoV‑2 or Mycobacterium tuberculosis ), client laboratories must execute a verified chemical and heat lysis inactivation protocol (e.g., 4% SDS buffer heated to 95°C for 10–15 min, or validated chaotropic guanidine lysis) prior to shipping. Samples must be certified non‑infectious before entering our mass spectrometry workflow.

Q: How do you perform database searching without inflating the False Discovery Rate (FDR) across two species?

Searching dual‑species proteomes using a merged database can lead to cross‑species peptide matching and distorted FDR calculations. Our bioinformatic pipeline utilizes concatenated FASTA databases with separate target‑decoy sub‑databases for the host (e.g., human or mouse) and the pathogen (e.g., bacterial or viral strain). We compute species‑specific peptide and protein FDR thresholds (strictly ≤1%), ensuring that low‑abundance pathogen identifications are statistically rigorous and not compromised by host spectral dominance.

Dual-Species Resolution · Host Immune Defense · Virulence Factor Kinetics · 4D-DIA Proteomics

During infectious disease progression, host cells mount rapid innate immune defense and metabolic rewiring, while pathogens inject dedicated virulence factors to subvert cellular machinery. Creative Proteomics provides a specialized Host–Pathogen Quantitative Proteomics Service using deep single-shot DIA, 4D ion-mobility mass spectrometry (dia-PASeF), and integrated dual-species bioinformatic pipelines to simultaneously quantify host immune signaling networks and pathogen virulence proteins without antibody dependence.

Simultaneous dual-species quantification: Co-identifies thousands of host proteins alongside low-abundance bacterial, viral, or fungal proteomes from a single infection lysate
Host innate immune signaling resolution: Captures early PRR, cGAS-STING, and NF-κB kinase cascades (0.5–2 h) alongside sustained interferon-stimulated gene (ISG) induction (12–48 h)
Pathogen virulence factor profiling: Quantifies bacterial secretion system effectors (T3SS, T4SS, T6SS), pore-forming toxins, and viral structural/non-structural complexes across the infection lifecycle
Rigorous biosafety & inactivation protocols: Standardized BSL-2-compliant lysis and heat-inactivation SOPs preserving complete peptide integrity while ensuring biological safety
Multi-pathogen platform versatility: Validated analytical workflows across viral models (SARS-CoV-2, Influenza, Flaviviruses), intracellular bacteria (M. tuberculosis, Salmonella, Listeria), and fungal pathogens (Candida, Aspergillus)

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What Is Host–Pathogen Quantitative Proteomics?

Host–pathogen quantitative proteomics is the simultaneous mass spectrometry-based profiling of host cellular defense networks and pathogen virulence factors within infected systems. Powered by high-depth DIA quantitative proteomics and 4D ion-mobility mass spectrometry, it resolves dynamic pathogen subversion and host defense kinetics across time courses and antimicrobial interventions.

While RNA-seq and antibody panels capture static transcriptional activation or isolated surface antigens, they fail to detect viral host shutoff, pathogen-mediated proteolysis, or pre-formed bacterial effector injection. Direct dual-species quantitative LC-MS/MS resolves the true functional proteome by quantifying thousands of host defense regulators alongside scarce pathogen effectors from a single infection lysate.

Primary Study Scenarios & Decision Nodes:

Viral Pathogenesis & Host Shutoff: Map viral non-structural proteins (e.g., RdRp, proteases) alongside host interferon cascades (cGAS-STING, ISG15, IFIT1–3) and translational arrest kinetics (0.5–24 h).
Intracellular Bacterial Vacuolar Escape: Quantify Type III/IV/VI secretion system effectors (T3SS/T4SS) and host immunometabolic switches (ACOD1/itaconate, Rab GTPases) across infected macrophages.
Virulence Attenuation & Knockout Profiling: Benchmark wild-type pathogens against isogenic Δvirulence or Δtoxin mutants to pinpoint direct cytotoxicity mechanisms and Caspase-1/Gasdermin D pyroptosis.
Antimicrobial & Antiviral Drug Deconvolution: Track dose-dependent suppression of pathogen structural complexes alongside the resolution of host pro-inflammatory cytokine storms.

Content Guide

Dual-Species Dynamic Range
Host vs. Pathogen Modules
Research Applications
Step-by-Step Workflow
Platform Decision Guide
Sample Submission
Deliverables & Outputs

Overcoming the Asymmetric Dynamic Range in Dual-Species Proteomics

The primary analytical bottleneck in host–pathogen proteomics is severe biomass asymmetry: host proteins frequently account for >95–99% of total sample protein, while pathogen proteins represent less than 1–5% of the total mass spectrometry signal. Creative Proteomics deploys a multi-tiered analytical strategy to maximize pathogen peptide recovery without sacrificing host depth:

Dual-species quantitative proteomics extraction workflow

Subcellular & Phagosomal Enrichment: For intracellular pathogens, sucrose gradient fractionation isolates intact phagosomes and host vacuolar membranes, enriching pathogen-associated factors by 10- to 30-fold before lysis.
Selective Microbial Pellet Recovery: Differential centrifugation combined with mild non-ionic detergent washes selectively lyses host plasma membranes while preserving intact bacterial cell walls for enriched microbial profiling.
High-Capacity 4D-MS Separation: Trapped ion mobility spectrometry (TIMS) on the timsTOF Pro 2 separates co-eluting, high-abundance host peptides from low-abundance pathogen ions in the collision cross-section (CCS) dimension, drastically increasing spectral purity.
Concatenated Dual-Species Database Searching: Joint search of host and pathogen FASTA databases with independent target-decoy calculation and strict false discovery rate (FDR ≤ 1%) controls eliminates cross-species mismatch artifacts.

Dual-Layer Functional Modules: Host Defense vs. Pathogen Virulence

Proteins quantified across infection cohorts are systematically clustered into coordinated host-response and pathogen-effector modules:

1. Host Innate Immunity & PRRs

Pattern recognition receptors (TLR2/4, cGAS-STING, RIG-I), adaptors (MyD88, TRIF, MAVS), and classical interferon-stimulated factors (ISG15, IFIT1–3, MX1, OAS1–3). Explore pathogen-mediated Ubiquitination Rewiring.

2. Host Stress & Immunometabolism

ER stress/UPR effectors (BiP, CHOP), glycolytic switches (GLUT1, HK2, LDHA), and antibacterial metabolic enzymes (ACOD1/IRG1 itaconate biosynthesis).

3. Pathogen Virulence Machinery

Bacterial secretion system effectors (Type III, IV, VI effectors, e.g., Inv/Prg/Spi), pore-forming toxins, and viral replication-transcription complexes. Map surface Glycoprotein Interactions.

4. Pathogen Defense & Counter-Measures

Antioxidant enzymes (catalases, SodA/KatG), bacterial heat-shock chaperones (DnaK, GroEL), multi-drug efflux pumps, and host-targeted proteases.

Translational Research Applications: Virology, Bacteriology, and Drug Discovery

Application	Model Systems	Analytical Objective	Key Molecular Readouts
Viral Infection & Host Shutoff	SARS-CoV-2, Influenza A, Dengue/Zika, RSV in primary epithelial cells.	Map viral protein accumulation and host translational arrest.	Viral RdRp/nucleoproteins, ribosomal subunits, ISGs (MX1, IFIT1), phosphorylated IRF3.
Bacterial Vacuolar Escape	M. tuberculosis, Salmonella, Listeria in Primary Macrophages.	Track phagosomal maturation arrest and metabolic reprogramming.	Bacterial T3SS/T4SS effectors, Rab5/Rab7, LAMP1, ACOD1/itaconate enzymes, v-ATPase.
Virulence Factor Perturbation	Wild-type vs. Δvirulence mutant pathogens in co-culture models.	Identify direct cytotoxicity and host immune evasion pathways.	Caspase-1/3/8 activation, Gasdermin D cleavage, NF-κB target cytokines, pathogen fitness.
Antimicrobial Drug Screening	Infected in vitro / in vivo models treated with antibiotics or antivirals.	Measure pathogen clearance and Drug Mechanism of Action regarding inflammatory cytokine storms.	Pathogen structural protein degradation, reduction of pro-inflammatory cytokines, tissue repair.
Fungal Dimorphism & Invasion	Candida albicans, Aspergillus co-cultured with host endothelial cells.	Quantify adhesins driving yeast-to-hyphae morphological transition.	Hyphal adhesins (Als3, Hwp1), secreted aspartyl proteases (Saps), host tight-junction claudins.

Step-by-Step Host–Pathogen Proteomics Workflow

Study Scoping & Infection Design

Define biological cohorts (Mock vs. infected, time series 0–48 h, MOI titrations) with matched uninfected controls and ≥4 biological replicates.

Biosafety Validation & Inactivation

Execute verified chaotropic lysis (4% SDS / 8M Urea) and heat inactivation (95°C, 10–15 min) ensuring complete non-infectivity for BSL-2 compatibility.

Dual-Proteome S-Trap Digestion

Automated S-Trap micro-column processing for total detergent clearance, reduction/alkylation, and rapid high-efficiency tryptic digestion.

High-Depth LC-MS/MS Acquisition

Single-shot DIA on Orbitrap Astral or 4D-DIA (dia-PASeF on timsTOF Pro 2) with interleaved pooled QC injections to ensure quantitative stability.

Joint Dual-Species Database Search

Search concatenated host and pathogen FASTA databases with independent target-decoy calculation and strict species-specific FDR ≤ 1% filtering.

Integrative Biological Reporting

Deliver normalized dual-species expression matrices, host defense pathway enrichment, pathogen virulence kinetics, and targeted PRM validation lists.

Study Design

Cohort & MOI definition

Inactivation

Lysis & heat clearance

Digestion

S-Trap surfactant removal

DIA / 4D-MS

Astral / timsTOF acquisition

Dual Search

Concatenated FASTA & FDR

Final Report

Dual kinetics & GSEA

Simultaneous host & pathogen resolution: captures both defense cascades and virulence effectors in one run
Joint concatenated database searching: independent target-decoy calculation prevents cross-species FDR inflation
High-capacity 4D-MS ion mobility: separates co-eluting host peptides from low-abundance microbial ions
Standardized biosafety workflows: validated chemical and thermal inactivation protocols compliant with BSL-2 standards

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Host–Pathogen Proteomics Analytical Platform Decision Guide

Study Goal	Strategy	Primary Platform	Deliverable Scope
Global Infection Discovery (Bulk Cell Lysates / In Vivo Tissues)	Discovery DIA Proteomics	Orbitrap Astral / Exploris 480 / timsTOF Pro 2	Deepest proteome depth (>6,000–8,500 host proteins; hundreds of microbial pathogen proteins), CV < 15%, missing value rates < 5%.
Microscale & Sorted Pathogen Samples (FACS-Sorted Infected Cells / Organoid Foci)	High-Sensitivity 4D-DIA	timsTOF Pro 2 / timsTOF Ultra (dia-PASeF)	TIMS ion mobility separates scarce pathogen peptides from abundant host ions; sensitive down to 50,000 sorted cells.
Immediate PRR & Kinase Signaling (cGAS-STING, TBK1, NF-κB, 15–120 min)	4D Phosphoproteomics	timsTOF Pro 2 / Orbitrap Exploris 480	Captures >15,000–25,000+ phosphosites; KSEA maps immediate kinase activation cascades triggered by pathogen contact.
Targeted Virulence & Toxin Panels (T3SS Effectors, Viral Antigens, Toxins)	Targeted PRM Panels	Orbitrap PRM / Triple Quadrupole (TSQ Altis)	Absolute quantification of 20–50 curated virulence targets across drug cohorts with zero missing values using SIL peptides.
Polymicrobial & Microbiomes (Gut, Stool, Mucosal Flora)	4D Metaproteomics Services	timsTOF Pro 2 / Orbitrap Astral	Taxonomic classification and functional pathway profiling across complex uncultivated multi-species microbial communities without host reference dependencies.
Host Antigen Presentation (MHC-I / MHC-II Epitopes)	Immunopeptidome Profiling Service	Orbitrap Eclipse / Exploris 480 / timsTOF Ultra	Direct immunoaffinity isolation and mass spectrometry sequencing of pathogen-derived peptides physically presented by host HLA complexes.

Sample Submission Requirements and Guidelines

Sample Type	Recommended Input	Minimum Input	Inactivation & Shipping Guidelines
Infected Adherent Cells	2–5 × 10⁶ cells / pellet	1 × 10⁶ cells	Wash 2× with cold PBS; lyse in 4% SDS or 8M Urea; heat inactivate (95°C, 10 min); ship on dry ice (-80°C).
Infected Suspension / Sorting Pellets	2–5 × 10⁶ cells / pellet	5 × 10⁵ cells	Pellet cells (300 × g, 5 min); rinse with cold PBS; lyse in chaotropic buffer; heat inactivate; ship on dry ice.
Infected Animal Tissues (Lung, Liver)	30–50 mg wet weight	15 mg wet weight	Dissect rapidly; bead-beat in SDS lysis buffer in BSL-2 hood; heat inactivate before packaging; ship on dry ice.
Conditioned Media (Secretomes)	5–10 mL culture supernatant	2 mL	Clarify (1,000 × g, 10 min); filter via 0.22 µm membrane; concentrate via 3 kDa filter; ship on dry ice.
Pre-Extracted & Inactivated Lysates	50–100 µg total protein	20 µg total protein	Document buffer formulation and sterility verification in submission documentation; ship on dry ice.

Deliverables and Decision-Ready Outputs

Dual-Species Expression Matrices, Infection Kinetics, and Virulence Factor Scorecards

PCA clustering of mock, acute, and sustained infection cohorts

Sample-level PCA evaluates biological cohort separation across mock, acute, and sustained infection stages.

Dual-species temporal heatmap of host defense and pathogen virulence factors

Hierarchical clustering resolves coordinated host defense induction versus pathogen virulence factor kinetics.

Volcano plot of differential host and pathogen proteins

Dual-species volcano plots identify statistically significant host effectors and targeted pathogen virulence factors.

Innate immunity pathway and kinase-substrate signaling network

Integrated signaling networks map cGAS-STING, NF-κB, and microbial secretion effector interactions.

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Dual-Species Normalized Quantitative Matrices

Log2-transformed expression tables containing separate and concatenated host and pathogen abundance metrics with species-specific FDR ≤ 1% filtering.

Kinetic Clustering & Virulence Profiling

Dynamic trajectory clustering of secretion system effectors, pore-forming toxins, and host antiviral/antibacterial regulators across infection time courses.

Statistical Contrast & Volcano Filtering

Pairwise significance testing (adjusted p-value ≤ 0.05, fold change ≤ 2.0) comparing Mock vs. Infected, mutant strains, and antimicrobial rescue cohorts.

Innate Immunity Pathway & GSEA Reports

Gene Set Enrichment Analysis (GSEA) mapping cGAS-STING, Type I interferon, NF-κB, inflammasome activation, and immunometabolic rewiring cascades.

Host–Pathogen Quantitative Proteomics Frequently Asked Questions

How does quantitative mass spectrometry compare to RNA-seq for host–pathogen infection studies?

While RNA-seq measures transcriptional responses, it cannot evaluate post-transcriptional host shutoff, viral-induced ribosome degradation, or bacterial proteolysis of host defense proteins. Furthermore, RNA-seq cannot quantify physical virulence toxins, bacterial secretion effectors, or post-translational kinase signaling cascades (e.g., TBK1 or p38 phosphorylation). Quantitative mass spectrometry provides an unbiased readout of actual functional protein abundance and pathway rewiring in both the host and pathogen simultaneously.

How do you prevent high-abundance host proteins from masking low-abundance pathogen proteins?

We utilize a combination of pre-analytical and analytical strategies: (1) physical enrichment protocols, such as gentle differential centrifugation to pellet intact bacterial cells or organellar isolation of infected phagosomes; (2) high-resolution 4D-DIA mass spectrometry on the timsTOF Pro 2, where trapped ion mobility (TIMS) separates co-eluting host and pathogen peptide ions across the collisional cross-section (CCS) dimension; and (3) optimized microflow chromatography delivering exceptional peak capacity and high signal-to-noise ratios.

What biosafety levels (BSL) can your facility accommodate for host–pathogen samples?

Creative Proteomics accepts samples derived from BSL-1 and BSL-2 biological agents. For pathogens classified under BSL-3 (such as SARS-CoV-2 or Mycobacterium tuberculosis), client laboratories must execute a verified chemical and heat lysis inactivation protocol (e.g., 4% SDS buffer heated to 95°C for 10–15 min, or validated chaotropic guanidine lysis) prior to shipping. Samples must be certified non-infectious before entering our mass spectrometry workflow.

How do you perform database searching without inflating the False Discovery Rate (FDR) across two species?

Searching dual-species proteomes using a merged database can lead to cross-species peptide matching and distorted FDR calculations. Our bioinformatic pipeline utilizes concatenated FASTA databases with separate target-decoy sub-databases for the host (e.g., human or mouse) and the pathogen (e.g., bacterial or viral strain). We compute species-specific peptide and protein FDR thresholds (strictly ≤ 1%), ensuring that low-abundance pathogen identifications are statistically rigorous and not compromised by host spectral dominance.

Can host–pathogen proteomics quantify both the intracellular proteome and secreted virulence factors?

Yes. Our workflow supports both cell lysate analysis and conditioned cell culture media (secretome) profiling. For conditioned media, supernatants are collected under defined serum-free or low-serum conditions, filtered through 0.22 µm membranes to remove bacterial and host cell debris, and concentrated using 3 kDa molecular weight cut-off filters to quantify secreted bacterial toxins (e.g., hemolysins, enterotoxins) and host cytokine secretomes.

What experimental replicates and controls are required for robust statistical analysis?

Because primary biological responses to infection can exhibit variable kinetics, we strongly recommend a minimum of 4 biological replicates per condition (e.g., 4 uninfected Mock replicates, 4 infected replicates per time point or MOI). Essential controls include procedure-matched Mock controls (exposed to identical media and handling without pathogen) and, when evaluating drug treatments, vehicle-treated infected and uninfected controls.

Can you profile post-translational modifications (PTMs) during host–pathogen infection?

Yes. We offer specialized 4D Phosphoproteomics to map early kinase-substrate cascades (e.g., STING-TBK1-IRF3, MAPK, and NF-κB activation) or T-Cell Activation occurring within pathogen contact. Additionally, we provide custom workflows for ubiquitination and acetylation profiling to investigate pathogen-mediated immune evasion mechanisms.

What pathogen information must be provided prior to project initiation?

Clients must provide: (1) exact genus, species, and reference strain designation (with NCBI TaxID or UniProt Proteome ID); (2) biosafety level and verified inactivation documentation; (3) host cell type and organism (human, mouse, rat, non-human primate); (4) infection model parameters (MOI, exposure duration, harvest method); and (5) project focus (host immune discovery, pathogen virulence quantification, or dual-proteome joint profiling).

* For Research Use Only. Not for use in the treatment or diagnosis of disease.

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Please submit a detailed description of your project. We will provide you with a customized study plan to meet your requests. You can also send us an email to for inquiries.