Efficient removal of sodium dodecyl sulfate (SDS) enhances analysis of proteins by SDS-polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Feng, Li; Dong, Maoqing; Miller, Laurence J.; Naylor, Stephen

doi:10.1002/(sici)1097-0231(19990315)13:5<464::aid-rcm486>3.0.co;2-z

Cited by 20 publications

(15 citation statements)

References 26 publications

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“…Common ionic detergents, such as SDS, suppress the MS detection of peptides and proteins and should be generally washed out before the analysis [39–40]. Application of nonionic surfactants, e.g., octyl-β-glucopyranoside (OBG), allows improving the recovery of peptides by MALDI-MS analysis [40–41].…”

Section: Resultsmentioning

confidence: 99%

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

Sharov¹,

Galeva²,

Dremina³

et al. 2009

Archives of Biochemistry and Biophysics

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There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function (Sharov et al., Exp. Gerontol. 41, 407-416;; in another report, the nitration of one specific residue, Tyr 613 , located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation (Dairou et al., J. Mol. Biol. 372, 1009-1021. In the present study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr 613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite. KeywordsGlycogen phosphorylase b; Enzymatic activity; Tyrosine nitration; Cysteine oxidation; Peroxynitrite; Mass spectrometry; Solvent accessible surface area There is increasing evidence that reactive nitrogen species nitrate specific tyrosine residues in proteins, and that the formation of 3-nitrotyrosine (3-NT) causes functional changes in targeted proteins [1][2][3][4]. Biological nitration of protein tyrosine residues has been demonstrated in various diseases and biological aging [5][6][7][8][9][10][11][12][13]. Protein nitration appears to be a rather selective process since neither all tyrosine residues in proteins nor all proteins in a given proteome get nitrated both in vivo [6,14,15] and in vitro [15][16][17][18]. In addition, modifications of different Tyr residues may not be evenly important for protein function. Several sources of Tyr nitration in vivo have been established involving reactions of peroxynitrite and/or nitrogen dioxide, or Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [19]. Regardless of the chemical mechanism, tyrosine nitration appears to be a complex process depending on the individual reactivity of Tyr residues in a protein, environment (pH, concentrations of reagents, solvent accessibility and diffusion coefficients), tissue specific protection, protein repair and turnover mechanisms. The knowledge of sequence location and the yields of respective T...

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Section: Resultsmentioning

confidence: 99%

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

Sharov¹,

Galeva²,

Dremina³

et al. 2009

Archives of Biochemistry and Biophysics

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“…detection is performed after the entire separation procedure has been completed. The off-line approach involves subsequent but separate MALDI analysis of fractions collected from HPLC, gel permeation chromatography (GPC) and capillary electrophoresis (CE) or spots scraped and extracted from thin layer chromatograms (TLC) and polyacrylamide gels [135,136,137,138,139,140]. Meldal and coworkers [139] have demonstrated rapid identification of organic reaction products using off-line TLC MALDI.…”

Section: Separations -Maldimentioning

confidence: 99%

Small molecule analysis by MALDI mass spectrometry

2002

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This review focuses on the application of matrix assisted laser desorption/ionization (MALDI) mass spectrometry to the characterization of molecules in the low mass range (<1500 Da). Despite its reputation to the contrary, MALDI is a powerful technique to provide both qualitative and quantitative determination of low molecular weight compounds. Several approaches to minimize interference via sample preparation and matrix selection are discussed, as well as coupling of MALDI to liquid and planar chromatographic techniques to extend its range of applicability.

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“…Subsequent use of complementary isolation techniques based on ion exchange, isoelectric focusing or affinity chromatography was therefore not feasible. Methods for removal of SDS have been described that involved chromatographic separation [4][5][6], buffer equilibration [7] or precipitation [8,9] steps. These methods are not suitable for insoluble membrane proteins that tend to precipitate irreversibly and adhere to surfaces.…”

Section: Introductionmentioning

confidence: 99%

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: Application to the purification of the fusion protein of the human immunodeficiency virus type 1

2002

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We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.

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Efficient removal of sodium dodecyl sulfate (SDS) enhances analysis of proteins by SDS-polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cited by 20 publications

References 26 publications

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

Small molecule analysis by MALDI mass spectrometry

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: Application to the purification of the fusion protein of the human immunodeficiency virus type 1

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