A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

Sim, Kae Hwan; Liu, Lillian Chia-Yi; Tan, Hwee Hoon; Tan, Ker Sin; Ng, Daniel; Zhang, Wei; Yang, Yuan Sheng; Tate, Stephen; Bi, Xuezhi

doi:10.1038/s41597-020-00594-z

Cited by 22 publications

(27 citation statements)

References 42 publications

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“…Label‐free SWATH‐MS proteomics was used to determine the effect of cell age on HCP composition. This DIA method for LC‐MS/MS enables high‐throughput and reproducible quantitation of diverse protein species within complex samples like HCCF (Sim et al, 2020). SWATH‐MS has been previously leveraged to characterize CHO cell lines and bioprocesses by tracking the levels of individual HCPs (Orellana et al, 2015; Walker et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

Hamaker

Min

Lee

2022

Biotech & Bioengineering

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The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industrywide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis.More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.

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Section: Resultsmentioning

confidence: 99%

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

Hamaker

Min

Lee

2022

Biotech & Bioengineering

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“…Since the early days of DIA, a high-quality spectral library either constructed experimentally or computationally has been considered a prerequisite for good spectral matching in the DIA analysis. As such, a lot of effort has been put towards building a comprehensive library via extensive DDA analyses [ 15 , 16 , 32 , 33 ]. There are various studies showing that a sample specific library results in better identification [ 34 , 35 ], thus justifying the resources required for library generation.…”

Section: Discussionmentioning

confidence: 99%

Narrow Precursor Mass Range for DIA–MS Enhances Protein Identification and Quantification in Arabidopsis

Zhang

Bensaddek

2021

Life

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Data independent acquisition–mass spectrometry (DIA–MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA–MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on an Arabidopsis sample with spike-in known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400–1200 m/z. When combining several DIA–MS analyses with narrow precursor ranges (i.e., 400–650, 650–900 and 900–1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 54.7% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the six-protein mix spike-in. In cDIA only five out of the six-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

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“…Since the early days of DIA, a high-quality spectral library either constructed experimentally or computationally has been considered a pe-requisite for good spectral matching in the DIA analysis. As such, a lot of effort has been put towards building a comprehensive library via extensive DDA analyses [15,16,31,32]. There are various studies showing that the sample specific library results in better identifications [33,34] thus justifying the resources required for library generation.…”

Section: Discussionmentioning

confidence: 99%

Narrow Precursor Mass Range for DIA-MS Enhances Protein Identification and Quantification

Zhang¹,

Bensaddek²

2021

Preprint

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Data independent acquisition - mass spectrometry (DIA-MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA-MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on Arabidopsis sample with spike-in of known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400-1200 m/z. When combining several DIA-MS analyses with narrow precursor ranges (i.e., 400-650, 650-900 and 900-1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 59.4% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the 6-protein mix spike-in. In cDIA only 5 out of the 6-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

show abstract

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins

Cited by 22 publications

References 42 publications

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

Narrow Precursor Mass Range for DIA–MS Enhances Protein Identification and Quantification in Arabidopsis

Narrow Precursor Mass Range for DIA-MS Enhances Protein Identification and Quantification

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