Enzyme-Friendly, Mass Spectrometry-Compatible Surfactant for In-Solution Enzymatic Digestion of Proteins

Yu, Ying-Qing; Gilár, Martin; Lee, Peter J.; Bouvier, Edouard S. P.; Gebler, John C.

doi:10.1021/ac0346196

Cited by 309 publications

(319 citation statements)

References 25 publications

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“…It is common practice to employ techniques to reduce incubation times, while retaining high sequence coverage and enzymatic specificity. Methods have included addition of organic solvents such as acetonitrile or methanol, detergent (urea), or acid-labile surfactants (RapiGest TM ) to digestion buffers to increase the digestion efficiencies and decrease incubation times (Russell et al, 2001;Yu et al, 2003;Umar et al, 2005). Other methods employed to decrease proteolytic incubation times have included the use of immobilized enzymes (Massolini & Calleri, 2005;Duan et al, 2006), and the immobilization of proteins to PVDF membrane followed by incubation with non-ionic surfactants and proteolytic enzymes (Pham, Henzel, & Lill, 2005).…”

Section: A Proteolytic Enzymesmentioning

confidence: 99%

Microwave‐assisted proteomics

Lill¹,

Ingle²,

Liu³

et al. 2007

Mass Spectrometry Reviews

145

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State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instrumentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characterization workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectrometric characterization. Techniques for microwave-assisted biocatalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of posttranslational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied. #

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Section: A Proteolytic Enzymesmentioning

confidence: 99%

Microwave‐assisted proteomics

Lill¹,

Ingle²,

Liu³

et al. 2007

Mass Spectrometry Reviews

145

View full text Add to dashboard Cite

show abstract

“…A common practice is to adopt methods capable of reducing incubation times, while retaining high sequence coverage and enzymatic specificity. Methods of improving digestion efficiency include thermal denaturation [1] and chemical denaturation (i.e., detergents or acid-labile surfactants or organic solvents) [2,3]. Other methods to decrease proteolytic incubation times include using immobilized enzymes [4].…”

mentioning

confidence: 99%

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Reddy

Hsu

et al. 2010

J. Am. Soc. Mass Spectrom.

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Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60°C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern. (J Am Soc Mass Spectrom 2010, 21, 421-424)

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“…Briefly, the samples were thermally denatured by incubation at 90°C for 20 min, while the chemical denaturation was performed using RapiGest TM SF (Waters) 3) . The samples solubilized in 0.1% RapiGest were incubated with 5 mM dithiothreitol (DTT) at 60°C for 30 min, followed by addition of iodoacetamide to 15 mM in the dark before incubation for 30 min.…”

Section: Denaturation and Tryptic Digestionmentioning

confidence: 99%

2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells: Combination with various sample preparation methods before in-solution trypsin digestion

et al. 2005

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SUMMARYTwo-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying lowmolecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.

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Enzyme-Friendly, Mass Spectrometry-Compatible Surfactant for In-Solution Enzymatic Digestion of Proteins

Cited by 309 publications

References 25 publications

Microwave‐assisted proteomics

Microwave‐assisted proteomics

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells: Combination with various sample preparation methods before in-solution trypsin digestion

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