Acid‐labile surfactant assists in‐solution digestion of proteins resistant to enzymatic attack

Suder, Piotr; Bierczynska, Anna; König, Simone; Silberring, Jerzy

doi:10.1002/rcm.1411

Cited by 25 publications

(17 citation statements)

References 3 publications

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“…This surfactant is anionic and has been reported to improve the solubilization/denaturation of proteins while maintaining the activity of trypsin. Trypsin maintains 100% of its activity in 0.1% RapiGest and improves the digestion of proteins commonly resistant to proteoly-sis (19). A secondary advantage of RapiGest is that upon acid treatment it is cleaved into two components, a small ionic moiety and a hydrophobic, aqueous insoluble group.…”

Section: Digestion Protocols Using Rapigest Have Improved Protein Idementioning

confidence: 99%

Effects of Modified Digestion Schemes on the Identification of Proteins from Complex Mixtures

2006

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In shotgun proteomics, a complex protein mixture is digested to peptides, separated and identified by microcapillary liquid chromatography followed by tandem mass spectrometry (LC-MS-MS). In this technology, complete protein digestion is often assumed. We show that, to the contrary, modifications to a standard digestion protocol demonstrate large, reproducible improvements in protein identification, a result consistent with digestion being a limiting factor in the efficiency of protein identification.

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Section: Digestion Protocols Using Rapigest Have Improved Protein Idementioning

confidence: 99%

Effects of Modified Digestion Schemes on the Identification of Proteins from Complex Mixtures

2006

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“…Myoglobin, ubiquitin, and bacteriorhodopsin were selected in this study because they have been reported to be moderately or highly resistant to tryptic digestion (18). The proteins were subjected to Tube-Gel digestion in the presence of SDS, Tube-Gel digestion in the absence of SDS, or in-solution digestion with SDS.…”

Section: Resultsmentioning

confidence: 99%

“…It was unclear how efficiently the authors were able to eliminate the SDS. An acid-labile detergent was recently reported to be able to assist in solubilizing membrane proteins and then degrade spontaneously when the sample is acidified (18). The usefulness of the acid-labile detergent remains to be further evaluated.…”

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confidence: 99%

Tube-Gel Digestion

Zhu

2005

Molecular & Cellular Proteomics

176

125

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This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl ␤-D-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. En- Membrane proteins participate in many important biological processes including cell adhesion, signal transduction, nutrient uptake, transportation, and endocytosis (1-3). There is an increasing level of interest in comprehensive and high throughput analysis of membrane proteins (4 -8). MS-based proteomics has rapidly developed in recent years as a powerful approach for the large scale identification and characterization of proteins. The commonly used bottom-up proteomic approach involves proteolytic digestion of proteins to peptides followed by mass spectrometric measurement of peptide masses and sequences (9 -14). Both MS and tandem MS data are subsequently subjected to database search for identification of unknown proteins and protein post-translational modifications. The confidence of protein identification is highly dependent on the number of peptides derived from each protein, which is determined by the efficiency of the proteolytic digestion as well as the coverage and accuracy of subsequent mass spectrometric analysis. However, there are substantial technical difficulties associated with both steps when analyzing membrane proteins. The proteolytic digestion of membrane proteins usually results in low sequence coverage because of their low solubility and high hydrophobicity, which render the cleavage sites less accessible to a proteolytic agent. Detergents and organic solvents can be used to improve the solubility of membrane proteins, but they interfere with subsequent MS measurement, particularly LC-MS/MS analysis, and generate poor MS data.Several methods have been reported for the solubilization and digestion of membrane proteins. Blonder et al. (15) used a high percentage (e.g. 60%) of methanol for the solubilization of Halobacterium purple membrane. Trypsin, a proteolytic enzyme commonly used in proteomics, retained ϳ20% activity in 60% methanol and was reported to be able to effectively digest bacteriorhodopsin, one of the major components of the purple membrane. Washburn et al. (16) tested the combination of ...

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“…The modification included incubation at a higher temperature (52°C) for a short period of time (3 min) and the use of a thermocycler (Applied Biosystems) as described originally by Turapov et al (48) and later by Moura et al (49). Briefly, a volume of 10 l of a 0.1% solution of sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-1-propanesulfonate (Rapigest, Waters Corporation, Milford, MA) in 50 mM ammonium bicarbonate digestion buffer was added to each 20 l phage aliquot to solubilize proteins and facilitate protein digestion (50,51). The solution was then incubated at 100°C for 5 min and rapidly cooled to room temperature.…”

Section: Preparation Of S Aureus Working Stock and Standard Solutionmentioning

confidence: 99%

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

Pierce

Rees

Fernández

et al. 2012

Molecular & Cellular Proteomics

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A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled 15 N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the 15 N (input phage) and 14 N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled 15 N bacteriophage peptide internal standard with that of the wild-type 14 N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 ؋ 10 4 colony forming units (CFU) ml ؊1 to 2.0 ؋ 10 6 CFU ml ؊1 , with the 15 N reference phage spiked at a concentration of 1.0 ؋ 10 9 plaque forming units (PFU) ml ؊1 . Amplification with 15 N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 ؋ 10 4 CFU ml ؊1

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Acid‐labile surfactant assists in‐solution digestion of proteins resistant to enzymatic attack

Cited by 25 publications

References 3 publications

Effects of Modified Digestion Schemes on the Identification of Proteins from Complex Mixtures

Effects of Modified Digestion Schemes on the Identification of Proteins from Complex Mixtures

Tube-Gel Digestion

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

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