Nonspecific cleavages arising from reconstitution of trypsin under mildly acidic conditions

Niu, Ben; Martinelli, Michael J.; Jiao, Yang; Wang, Chunlei; Cao, Mingyan; Wang, Jihong; Meinke, Eric

doi:10.1371/journal.pone.0236740

Cited by 16 publications

(14 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Besides, it has been shown to be related to a number of digestion conditions and predigestion treatment. − In this work, to prevent the autolysis of trypsin before spray, trypsin was reconstituted in 0.1% acetic acid. Although mildly acidic environment was often recommended by the manufacturer to prevent trypsin autolysis, a significantly increased level of nonspecific cleavages during the trypsin digestion process has been reported before …”

Section: Resultsmentioning

confidence: 99%

“…Although mildly acidic environment was often recommended by the manufacturer to prevent trypsin autolysis, a significantly increased level of nonspecific cleavages during the trypsin digestion process has been reported before. 67 In addition, we quantitatively compared each fully cleaved peptide based on the peak intensities reported from Maxquant. Interestingly, the abundance of different fully cleaved tryptic peptides was found to be quite distinct under the two digestion conditions.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

Gunawardena

et al. 2022

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 μs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of β-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

Gunawardena

et al. 2022

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Although using lower concentrations increased the number of PSMs identified in a shotgun run, incomplete or unexpected digestion products were observed in >25% of the results at the lowest concentration. Such low digestion efficiencies may prove beneficial when attempting to increase protein coverage but can be detrimental to analyses used for protein quantification. , …”

Section: Results and Discussionmentioning

confidence: 99%

“…Such low digestion efficiencies may prove beneficial when attempting to increase protein coverage but can be detrimental to analyses used for protein quantification. 25,26 SPACEPro also provides metrics to evaluate the peptidelevel digestion efficiency. Peptide analysis combines all PSMs into unique precursor signals based on the amino acid sequence and charge state.…”

Section: Analysis Of Enzyme Concentrationmentioning

confidence: 99%

SPACEPro: A Software Tool for Analysis of Protein Sample Cleavage for Tandem Mass Spectrometry

et al. 2021

View full text Add to dashboard Cite

The efficiency of shotgun proteomic analysis is dependent on the reproducibility of the peptide cleavage process during sample preparation. To generate rapid and useful metrics for peptide cleavage efficiency, as in enzymatic or chemical cleavage, SPACEPro was developed to evaluate efficiency and reproducibility of protein cleavage in peptide samples following data-dependent analysis by mass spectrometry. SPACEPro analyzes samples at the peptide-spectrum match (PSM), peptide, and protein levels to provide a comprehensive representation of the overall sample processing to peptides. All output is provided in human-readable text and JSON files that can be further processed to assess the cleavage efficiency on proteins within the sample. SPACEPro provides a snapshot of the protein cleavage efficiency through very minimal effort so that the user is informed of the quality of the sample processing efficiency and can accordingly develop protocols to improve the initial sample preparation for subsequent analyses.

show abstract

“…Several strategies have been explored to minimize disulfide scrambling, such as excess iodoacetamide (IAM) to cap all free sulfhydryl, enzymatic digestion at acidic pH using AccuMAP digestion kit, N-ethylmaleimide as an alkylating agent, and cystamine as the oxidizing agent. However, non-specific cleavage with poor digestion specificity and efficiency has been mreported, failing to prevent non-native disulfide formation [23][24][25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Elucidating chemical and disulfide heterogeneities in rituximab using reduced and non‐reduced peptide mapping

Nupur

Rathore

2022

J of Separation Science

View full text Add to dashboard Cite

Peptide mapping by liquid chromatography‐mass spectrometry is the gold standard to characterize post‐translational modifications (PTMs) and disulfide bonds. The structural integrity, heterogeneity, and quality of biotherapeutic proteins are evaluated via reduced and non‐reduced peptide mapping methods. However, non‐enzymatic artifacts are often induced during sample preparation when alkaline pH conditions are used. To minimize these artifacts, methods using various acidic pH conditions have been developed by multiple researchers. However, these may lead to missed and non‐specific cleavages during the analysis. In this study, an improved reduced and non‐reduced peptide mapping method has been proposed to characterize endogenous chemical modifications and native disulfide bonds of monoclonal antibody‐based products. Solubilization has been carried out at acidic pH conditions under high temperature, followed by the addition of tris (2‐carboxyethyl) phosphine as a reducing agent and a low alkylating agent. It was observed that the non‐enzymatic PTMs and non‐native disulfide scrambled peptides significantly reduced under trypsin plus endoproteinase Lys‐C digestion conditions at acidic pH as compared to the traditional methods. The results demonstrate that the proposed reduced and non‐reduced peptide mapping method using trypsin plus Lys‐C could identify and quantify various chemical and disulfide heterogeneities with minimal artifacts.

show abstract

Nonspecific cleavages arising from reconstitution of trypsin under mildly acidic conditions

Cited by 16 publications

References 34 publications

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution

SPACEPro: A Software Tool for Analysis of Protein Sample Cleavage for Tandem Mass Spectrometry

Elucidating chemical and disulfide heterogeneities in rituximab using reduced and non‐reduced peptide mapping

Contact Info

Product

Resources

About