Dataset Information

Surfactant-Induced Artifacts during Proteomic Sample Preparation.

ABSTRACT: Bottom-up proteomics is a powerful tool for characterization of protein post-translational modifications (PTMs), where PTMs are identified at the peptide level by mass spectrometry (MS) following protein digestion. However, enzymatic digestion is associated with additional sample processing steps that may potentially introduce artifactual modifications. Here, during an MS study of the PTMs of the regulator of G-protein signaling 4, we discovered that the use of ProteaseMAX, which is an acid-labile surfactant commonly used to improve protein solubilization and digestion efficiency, can lead to in vitro modifications on cysteine residues. These hydrophobic modifications resemble S-palmitoylation and hydroxyfarnesylation, thus discouraging the use of ProteaseMAX in studies of lipid modifications of proteins. Furthermore, since they target the cysteine thiol group, the presence of these artifacts will inevitably lead to inaccuracies in quantitative analysis of cysteine modifications.

SUBMITTER: Ji Y

PROVIDER: S-EPMC4574305 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Surfactant-Induced Artifacts during Proteomic Sample Preparation.

Ji Yuhuan Y Liu Minjing M Bachschmid Markus M MM Costello Catherine E CE Lin Cheng C

Analytical chemistry 20150520 11

Bottom-up proteomics is a powerful tool for characterization of protein post-translational modifications (PTMs), where PTMs are identified at the peptide level by mass spectrometry (MS) following protein digestion. However, enzymatic digestion is associated with additional sample processing steps that may potentially introduce artifactual modifications. Here, during an MS study of the PTMs of the regulator of G-protein signaling 4, we discovered that the use of ProteaseMAX, which is an acid-labi ...[more]

PMID: 25945600

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Project description:Background:Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol. Methods:Four single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition. Results:On average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC-MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%. Conclusions:In essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research.

Dataset Information

Surfactant-Induced Artifacts during Proteomic Sample Preparation.

Publications

Surfactant-Induced Artifacts during Proteomic Sample Preparation.

Similar Datasets

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