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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Divyanshi Karmani

LAB HEAD: John Patrick Murphy

PROVIDER: PXD050183 | Pride | 2026-01-12

REPOSITORIES: Pride

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			Action	DRS
	ea09142_0BSA_MS2_A.mzML	Mzml
	ea09142_0BSA_MS2_A.pepXML	Pepxml
	ea09142_0BSA_MS2_A.raw	Raw
	ea09144_0BSA_SPS_A.mzML	Mzml
	ea09144_0BSA_SPS_A.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 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.

2026-01-12 | PXD049970 | 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

Systematic analysis of non programmed frameshift suppression in E. coli via translational tiling proteomics

Project description:Programmed ribosomal frameshifting is a process that augments the coding capacity of particular mRNA transcripts. Programmed ribosomal frameshifting may occur during the decoding of chromosomally encoded genes in both prokaryotes and eukaryotes. Although programmed ribosomal frameshifting has been intensively studied, less is known about the occurrence of translation errors that result in frameshifts. Detection of unexpectedly efficient frameshift suppression in E. coli (at levels ranging from 1% to 10% with two unrelated test proteins) led us to investigate the scope of events responsible for restoration of the native frame. To enable a comprehensive approach, we developed a targeted mass-spectrometry method–“translational tiling proteomics”–allowing interrogation of the full set of possible –1 slippage events that could lead to the observed instances of frameshift suppression in vitro and in cells. Our findings point to an unexpectedly high basal level of ribosomal frameshifting, suggest that frameshifted products may contribute more significantly to the proteome than generally assumed, and raise the possibility that particular pseudogenes may specify functional proteins.

2024-01-11 | PXD047675 | Pride

cDNA microarray assessment of early gene expression profiles in Escherichia coli cells exposed to a mixture of heavy metals

Project description:Our main objective was to study the changes in cDNA microarray gene expression profiles of E. coli cells exposed to different doses of a polymetallic solution containing Ag (I), Pb (II), Cd (II), Cu (II), Ni (II) and Zn (II)) over three exposure times (5, 10 and 15 minutes). Control cells grown in the absence of metals were also included in the experiment.

2013-10-31 | E-MTAB-2023 | biostudies-arrayexpress

BS-CP: Efficient streaming Bayesian tensor decomposition method via assumed density filtering.

Project description:Tensor data is common in real-world applications, such as recommendation system and air quality monitoring. But such data is often sparse, noisy, and fast produced. CANDECOMP/PARAFAC (CP) is a popular tensor decomposition model, which is both theoretically advantageous and numerically stable. However, learning the CP model in a Bayesian framework, though promising to handle data sparsity and noise, is computationally challenging, especially with fast produced data streams. The fundamental problem addressed by the paper is mainly tackles the efficient processing of streaming tensor data. In this work, we propose BS-CP, a quick and accurate structure to dynamically update the posterior of latent factors when a new observation tensor is received. We first present the BS-CP1 algorithm, which is an efficient implementation using assumed density filtering (ADF). In addition, we propose BS-CP2 algorithm, using Gauss-Laguerre quadrature method to integrate the noise effect which shows better empirical result. We tested BS-CP1 and BS-CP2 on generic real recommendation system datasets, including Beijing-15k, Beijing-20k, MovieLens-1m and Fit Record. Compared with state-of-the-art methods, BS-CP1 achieve 31.8% and 33.3% RMSE improvement in the last two datasets, with a similar trend observed for BS-CP2. This evidence proves that our algorithm has better results on large datasets and is more suitable for real-world scenarios. Compared with most other comparison methods, our approach has demonstrated an improvement of over 10% and exhibits superior stability.

| S-EPMC11611110 | biostudies-literature

Gene expression in Escherichia coli treated with human PGRP, gentamicin, and CCCP

Project description:Human Peptidoglycan Recognition Proteins (PGRPs) kill bacteria, likely by over-activating stress responses in bacteria. To gain insight into the mechanism of PGRP killing of Escherichia coli and bacterial defense against PGRP killing, gene expression in E. coli treated with a control protein (bovine serum albumin, BSA), human recombinant PGRP (PGLYRP4), gentamicin (aminoglycoside antibiotic), and CCCP (membrane potential decoupler) were compared. Each treatment induced unique and somewhat overlapping pattern of gene expression. PGRP highly increased expression of genes for oxidative and disulfide stress, detoxification and efflux of Cu, As, and Zn, repair of damaged proteins and DNA, methionine and histidine synthesis, energy generation, and Fe-S clusters repair. PGRP also caused marked decrease in the expression of genes for Fe uptake and motility. Gene expression microarray in E. coli exposed to a human bactericidal innate immunity protein, PGRP, showed induction of oxidative stress response and defense genes, with different expression pattern than E. coli exposed to an aminoglycoside antibiotic and a membrane potential decoupler. E. coli was treated with PGRP (human recombinant PGLYRP4), gentamicin, or CCCP (carbonyl cyanide 3-chlorophenylhydrazone). RNA was obtained from each culture and assayed for gene expression using whole genome Affymetrix E. coli Genome 2.0 Array. Each experiment was repeated 3 times.

2014-07-25 | E-GEOD-44211 | biostudies-arrayexpress

Extensive Peptide Fractionation and y1 Ion-based Interference Detection Enable Accurate Quantification by Isobaric Labeling and Mass Spectrometry

Project description:Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. We pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ~20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. The quantitative interference was systematic investigated and the interference in MS2 scans were estimated by the intensity of contaminated y1 product ions. An algorithm was thus developed to correct reporter ion ratios of tryptic peptides.

2022-02-28 | PXD005797 | Pride

Expression data from the mosquito Anopheles gambiae midgut 3h after a protein meal with or without E. coli.

Project description:The Anopheles gambiae midgut harbors bacteria that proliferate upon a blood feed. We used microarrays to examine the midgut gene expression response at early stages (3hours) after an artifitial meal containing heat killed bacteria. Anopheles gambiae G3 mosquitoes 5-6 day-old were fed BSA (20% in PBS with fresh 10 mM sodium bicarbonate) with or without heat killed E. coli (equivalent of 2.5 ml of 0.8 OD) . Three pools of 10 mosquito midguts were dissected after 3h and processed for microarray analysis of gene expression.

2010-02-21 | E-GEOD-20204 | biostudies-arrayexpress