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Combine and conquer: surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins.

Waas M, Bhattacharya S, Chuppa S, Wu X, Jensen DR, Omasits U, Wollscheid B, Volkman BF, Noon KR, Gundry RL - Anal. Chem. (2014)

Bottom Line: Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach.A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage.Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

View Article: PubMed Central - PubMed

Affiliation: Milwaukee School of Engineering , Milwaukee, Wisconsin 53202, United States.

ABSTRACT
Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

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Summary of results fromphase I (individual additives). For each tube set, (A) the total numberof proteins identified, (B) number of unique peptide sequences identified,(C) average sequence coverage, and (D) number of peptides with hydrophobicGRAVY scores are shown. In (A) and (B), values for each biologicalreplicate are plotted separately to illustrate consistent overalltrends.
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fig2: Summary of results fromphase I (individual additives). For each tube set, (A) the total numberof proteins identified, (B) number of unique peptide sequences identified,(C) average sequence coverage, and (D) number of peptides with hydrophobicGRAVY scores are shown. In (A) and (B), values for each biologicalreplicate are plotted separately to illustrate consistent overalltrends.

Mentions: Two technical replicates of each samplewere analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS)on an LTQ linear ion trap (Thermo) as described in the Supporting Information. For all analyses summarizedin Figure 2–4, the resultswere based on the fully tryptic digest search. A separate databasesearch was conducted as described in SupportingInformation, but against a semitryptic peptide database, andwas used to assess the total number and percentage of spectra matchedto semitryptic peptides (i.e., only a single tryptic terminus), assummarized in Figure 5. Finally, to assessthe ability for each digestion condition to access peptides that spanthe predicted TM domains, a custom software tool was developed tomap identified peptides onto TM topology information curated in UniProt,which is a combination of experimentally determined information andpredictions, utilizing the predictive tools TMHMM, Memsat, Phobius,and hydrophobic moment plot method.13 Themapping software, PeptideEclipse, is open source and can be accessedat http://ulo.github.io/PeptideEclipse/. Within these studies,a TM peptide is defined as a peptide that contains at least one aminoacid from the annotated TM domain from UniProt.


Combine and conquer: surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins.

Waas M, Bhattacharya S, Chuppa S, Wu X, Jensen DR, Omasits U, Wollscheid B, Volkman BF, Noon KR, Gundry RL - Anal. Chem. (2014)

Summary of results fromphase I (individual additives). For each tube set, (A) the total numberof proteins identified, (B) number of unique peptide sequences identified,(C) average sequence coverage, and (D) number of peptides with hydrophobicGRAVY scores are shown. In (A) and (B), values for each biologicalreplicate are plotted separately to illustrate consistent overalltrends.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4007983&req=5

fig2: Summary of results fromphase I (individual additives). For each tube set, (A) the total numberof proteins identified, (B) number of unique peptide sequences identified,(C) average sequence coverage, and (D) number of peptides with hydrophobicGRAVY scores are shown. In (A) and (B), values for each biologicalreplicate are plotted separately to illustrate consistent overalltrends.
Mentions: Two technical replicates of each samplewere analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS)on an LTQ linear ion trap (Thermo) as described in the Supporting Information. For all analyses summarizedin Figure 2–4, the resultswere based on the fully tryptic digest search. A separate databasesearch was conducted as described in SupportingInformation, but against a semitryptic peptide database, andwas used to assess the total number and percentage of spectra matchedto semitryptic peptides (i.e., only a single tryptic terminus), assummarized in Figure 5. Finally, to assessthe ability for each digestion condition to access peptides that spanthe predicted TM domains, a custom software tool was developed tomap identified peptides onto TM topology information curated in UniProt,which is a combination of experimentally determined information andpredictions, utilizing the predictive tools TMHMM, Memsat, Phobius,and hydrophobic moment plot method.13 Themapping software, PeptideEclipse, is open source and can be accessedat http://ulo.github.io/PeptideEclipse/. Within these studies,a TM peptide is defined as a peptide that contains at least one aminoacid from the annotated TM domain from UniProt.

Bottom Line: Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach.A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage.Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

View Article: PubMed Central - PubMed

Affiliation: Milwaukee School of Engineering , Milwaukee, Wisconsin 53202, United States.

ABSTRACT
Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

Show MeSH