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Carrier protein-enabled host cell proteome analysis (CP-HCP) with integrated isobaric tag interference filtering

ABSTRACT: We used TMT labelling to set up different experiment channels containing mixtures of the host cell carrier proteome (E coli) and mock biotherapeutic (BSA), to compare with interference detection channel to model and filter interference, a phenomenon where host cell proteins (HCPs) co-elute with high-background biotherapeutic during LC-MS monitoring of QC during biomanufacture of biopharmaceuticals. Our work overcomes limitations of ELISA and pre-tryptic digest procedures to produce a reproducible method using TMT labels to identify and quantify a range of HCPs in one experiment.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Divyanshi Karmani

LAB HEAD: John Patrick Murphy

PROVIDER: PXD049970 | Pride | 2026-01-12

REPOSITORIES: Pride

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			Action	DRS
	hia.mzML	Mzml
	hia.pepXML	Pepxml
	hia.raw	Raw
	sht.mzML	Mzml
	sht.pepXML	Pepxml

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Publications

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Karmani Divyanshi D Seifihesar Niloofar N Sultonova Mukhayyo M Blackmore Beau B Paulo Joao A JA Harty Matthew M Murphy J Patrick JP

Journal of proteome research 20241009 11

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. T ...[more]

PMID: 39382319

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Carrier protein-enabled host cell proteome analysis (CP-HCP) with integrated isobaric tag interference filtering

Project description:We added increasing amounts of E coli carrier proteome to BSA, our moch recombinant protein to model interference of high-background BSA peaks with smaller peaks of E coli HCPs, using MS2 and MS3 methods to show co-fragmentation and co-elution based on similar m/z ratio. We created an interference detection channel to solve the problem of interference.

2026-01-12 | PXD050183 | Pride

Carrier protein-enabled host cell proteome analysis (CP-HCP) with integrated isobaric tag interference filtering.

Project description:Here, we proposed that the use of an HCP carrier proteome could enhance the acquisition of spectra from HCPs amidst a highly abundance therapeutic protein background to perform global HCP analysis. We provide proof of principle of this approach, which we term carrier proteome-HCP (CP-HCP) analysis, using a basic model of bovine serum albumin (BSA) as a therapeutic protein and BL21 expression strain E.coli as a host cell proteome. We observe that MS3 based methods do not alleviate interference in this model due to the high mock therapeutic background, so we added an interference detection channel to filter co-isolation interference-containing peptides. The approach also does not require any type of enrichment and can identify hundreds of E.coli peptides at small quantities amidst a high background protein.

2026-01-12 | PXD049386 | Pride

Carrier protein-enabled host cell proteome analysis (CP-HCP) with integrated isobaric tag interference filtering

Project description:We have created a CP-HCP method to characterise the carrier proteome of a host cell expression system producing recombinant protein biotherapeutic, which can be compared with the testing sample mixture containing trace amounts of host cell proteins against high-background recombinant protein and also a recombinant protein peptide characterisation channel, using TMT labelling.

2026-01-12 | PXD050214 | Pride

Multiplex secretome engineering enhances recombinant protein production and purity

Project description:Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

2020-03-19 | GSE144624 | GEO

USP28 enables oncogenic transformation of respiratory cells and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

Project description:Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and ∆NP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.

2022-04-08 | PXD032810 | Pride

SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Project description:Site-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and significantly lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search which increases the identification rates of intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

2020-08-24 | PXD018349 | Pride

A novel system for 3D co-culture hair follicle mimetic tissue

Project description:Our study developed a novel 3D co-culture system to mimic the in vivo ECM dynamics. Using this system, we examined the interaction between primary dermal papilla cells isolated from patients with neonatal keratinocytes.

2025-05-06 | PXD051461 | Pride

Analysis of functional connectivity changes from childhood to old age: A study using HCP-D, HCP-YA, and HCP-A datasets.

Project description:We present a new clustering-enabled regression approach to investigate how functional connectivity (FC) of the entire brain changes from childhood to old age. By applying this method to resting-state functional magnetic resonance imaging data aggregated from three Human Connectome Project studies, we cluster brain regions that undergo identical age-related changes in FC and reveal diverse patterns of these changes for different region clusters. While most brain connections between pairs of regions show minimal yet statistically significant FC changes with age, only a tiny proportion of connections exhibit practically significant age-related changes in FC. Among these connections, FC between region clusters from the same functional network tends to decrease over time, whereas FC between region clusters from different networks demonstrates various patterns of age-related changes. Moreover, our research uncovers sex-specific trends in FC changes. Females show much higher FC mainly within the default mode network, whereas males display higher FC across several more brain networks. These findings underscore the complexity and heterogeneity of FC changes in the brain throughout the lifespan.

| S-EPMC11894817 | biostudies-literature

Characterization of Primary Patient B-cells under Various Stimulation Conditions

Project description:The goal of this project was to compare the effects of differential stimulation of primary patient B-cells with CD40L, CpG, anti-IgM, and IL4 individually and in combination. Additionally, we observed a phenomenon where anti-IgM+CpG treatment resulted in a lymphoma-like phenotype and sought to compare this treatment regimen to the lymphoma cell line HBL-1.

2025-08-21 | PXD016961 | Pride

Pleiotropic effects of Metformin on the chemotherapy response of HPV-positive cancer cells

Project description:Metformin has been shown to downregulate viral E6/E7 expression in HPV-positive cancer cells. However, Metformin does not induce cellular senescence, which is observed upon E6/E7 repression by RNA interference. Here we compare mass spectrometry-based proteome analyses of Metformin-treated SiHa cells (previously deposited to the ProteomeXchange Consortium with data set identifier PXC011095) with proteome analyses of SiHa cells transfected with si16E6/E7 or control siRNA (siCtrl-1) and grown for 72 hours to evaluate differences in the cell cycle regulation of HPV-positive cancer cells upon E6/E7 repression by Metformin or siE6/E7.

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