Detergent Analysis in Protein Samples Using Mid‐Infrared (MIR) Spectroscopy

Chandreyee Das1, Timothy Nadler1, Ivona Strug1

1 EMD Millipore Corporation, Danvers, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.12
DOI:  10.1002/0471140864.ps2912s81
Online Posting Date:  August, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Quantitating relative levels of detergent present in protein preparations or samples derived from biological material, such as tissue or body fluids, is important because the presence of detergent may affect downstream analyses as well as protein structure/function. Especially because sample volumes, analysts’ available time, and other resources may be limited, a method that consumes little sample and that is rapid and simple is needed for detergent analysis. It would also be preferable to have a method that is generally applicable across many aliphatic chain‐containing molecules with many different physical properties. In this unit, methods are described for analyzing detergents and proteins in detergent‐protein mixtures using mid‐infrared (MIR) spectroscopy. A protocol is also included for efficient removal of unbound detergents from a protein sample accompanied by MIR‐based monitoring of both detergent and protein content. This rapid monitoring of sample preparation during the workflow enables users to make timely decisions about sample preparation strategies that maximize both analyte purity and yield. © 2015 by John Wiley & Sons, Inc.

Keywords: mid‐infrared (MIR) spectroscopy; infrared; detergent analysis; protein samples; detergent removal

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: General MIR Spectroscopy‐Based Analysis of Detergent‐Containing Samples
  • Basic Protocol 2: MIR Spectroscopy‐Based Analysis of Protein Content in a Complex Mixture Containing Both Protein and Detergent
  • Basic Protocol 3: MIR Spectroscopy‐Based Monitoring of Detergent Removal and Protein Recovery
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: General MIR Spectroscopy‐Based Analysis of Detergent‐Containing Samples

  Materials
  • PBS
  • Milli‐Q or other Type I Ultrapure water
  • Detergents and lysis buffers:
    • 2% sodium deoxycholate in PBS (store up to 24 hr at room temperature)
    • 4% sodium dodecyl sulfate (SDS) in PBS (store up to 6 months at room temperature)
    • 2% NP‐40 in PBS (store up to 1 year at 4°C)
    • 2% Triton X‐100 in PBS (store up to 1 year at 4°C)
    • 2× radioimmunoprecipitation assay (RIPA) lysis buffer (see recipe; store up to 6 months at 4°C)
  • Assay‐free sample cards (EMD Millipore, cat. no. DDAC00010‐8P)
  • Direct Detect MIR‐based spectrometer with most recent software version (EMD Millipore, cat. no. DDHW00010‐00)
  • P2, P20 and P200 micropipettors (e.g., Rainin)

Basic Protocol 2: MIR Spectroscopy‐Based Analysis of Protein Content in a Complex Mixture Containing Both Protein and Detergent

  Materials
  • PBS
  • Milli‐Q or other Type I Ultrapure water
  • Detergents and lysis buffer:
    • 2% sodium deoxycholate in PBS (store up to 24 hr at room temperature)
    • 2% NP‐40 in PBS (store up to 1 year at 4°C)
    • 2× radioimmunoprecipitation assay (RIPA) lysis buffer (see recipe; store up to 6 months at 4°C)
  • Bovine serum albumin (BSA) SRM927d (National Institute of Standards and Technology [NIST] certified; prepare solutions fresh and use immediately):
    • 10 mg/ml stock solution in PBS containing 0.5% sodium deoxycholate
    • 10 mg/ml stock solution in PBS containing 1% NP‐40
    • 10 mg/ml stock solution in 1× RIPA buffer
  • Cytochrome C (EMD Millipore, cat. no. 250600; store solutions 24 hr at 4°C):
    • 2 mg/ml in PBS containing 0.5% sodium deoxycholate
    • 2 mg/ml in PBS containing 1% NP‐40
  • ϒ‐globulins from rabbit (Sigma‐Aldrich, cat. no. G2018; store solutions 24 hr at 4°C):
    • 2 mg/ml in PBS containing 0.5% sodium deoxycholate
    • 2 mg/ml in PBS containing 1% NP‐40
  • Rat liver lysate (homogenize frozen rat liver in 1× RIPA buffer containing phosphatase and protease inhibitor cocktails; spin down several times by centrifuge; collect clear lysate solution and store 24 hr at 4°C)
  • Assay‐free sample cards (EMD Millipore, cat. no. DDAC00010‐8P)
  • Direct Detect spectrometer (EMD Millipore, cat. no. DDHW00010‐00)
  • P2, P20 and P200 micropipettors (e.g., Rainin)

Basic Protocol 3: MIR Spectroscopy‐Based Monitoring of Detergent Removal and Protein Recovery

  Materials
  • Sample to be analyzed (e.g., recombinant protein preparation solubilized in a sodium deoxycholate‐containing buffer; store up to 24 hr at 4°C)
  • 2% sodium deoxycholate in PBS (store up to 24 hr at room temperature)
  • PBS (store up to 1 year at room temperature)
  • Milli‐Q or other Type I Ultrapure water
  • ϒ‐globulins from rabbit (Sigma‐Aldrich, cat. no. G2018; 2 mg/ml in PBS containing 0.5% sodium deoxycholate; store up to 24 hr at 4°C)
  • Bovine serum albumin (BSA) SRM927d (National Institute of Standards and Technology [NIST] certified; 10 mg/ml stock solution in PBS containing 0.5% sodium deoxycholate; prepare solutions fresh and use immediately)
  • Amicon Pro Purification System (EMD Millipore)
  • Assay‐free sample cards (EMD Millipore, cat. no. DDAC00010‐8P)
  • Direct Detect spectrometer (EMD Millipore, cat. no. DDHW00010‐00)
  • P2, P20 and P200 micropipettors (e.g., Rainin)
  • Centrifuge with swinging bucket rotor (e.g., Beckman)
  • Microcentrifuge (e.g., Eppendorf)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Antharavally, B.S., Mallia, K.A., Rosenblatt, M.M., Salunkhe, A.M., Rogers, J.C., Haney, P., and Haghdoost, N. 2011. Efficient removal of detergents from proteins and peptides in a spin column format. Anal. Biochem. 416:39‐44. doi: 10.1016/j.ab.2011.05.013
  Barret, L.A., Polidori, A., Bonneté, F., Bernard‐Savary, P., and Jungas, C. 2013. A new high‐performance thin layer chromatography‐based assay of detergents and surfactants commonly used in membrane protein studies. J. Chromatogr. A 1281:135‐141.
  Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M., and Stefani, M. 2002. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416:507‐511.
  DaCosta, C.J. and Baenziger, J.E. 2003. A rapid method for assessing lipid: Protein and detergent: Protein ratios in membrane‐protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 59:77‐83.
  De Foresta, B., Henao, F., and Champeil, P. 1994. Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca2+‐ATPase by the non‐ionic detergent dodecylmaltoside. Eur. J. Biochem. 223:359‐369.
  Dreissig, I., Machill, S., Salzer, R., and Krafft, C. 2009. Quantification of brain lipids by FTIR spectroscopy and partial least squares regression. Spectrochim. Acta A Mol. Biomol. Spectrosc. 71:2069‐2075. doi: 10.1016/j.saa.2008.08.008
  EMD Millipore Corporation. 2012. Essential Biochemicals for Research: A technical resource and product guide. Literature Number PB3331EN00.
  EMD Millipore Corporation, 2014. Protocol: Detergent removal using Amicon Ultra or Microcon centrifugal ultrafilters. Literature Number PC5784EN00.
  Finley, J.B., Qiu, S.H., Luan, C.H., and Luo, M. 2004. Structural genomics for Caenorhabditis elegans: High throughput protein expression analysis. Protein Expr. Purif. 34:49‐55.
  Garavito, R.M. and Ferguson‐Miller, S. 2001. Detergents as tools in membrane biochemistry. J. Biol. Chem. 276:32403‐32406.
  Janatsch, G., Kruse‐Jarres, J.D., Marbach, R., and Heise, H.M. 1989. Multivariate calibration for assays in clinical chemistry using attenuated total reflection infrared spectra of human blood plasma. Anal. Chem. 61:2016‐2023.
  Kong, J. and Yu, S. 2007. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. (Shanghai) 39:549‐559.
  Miyazawa, T. and Blout, E.R. 1961. The infrared spectra of polypeptides in various conformations: Amide I and II bands. J. Am. Chem. Soc. 83:712‐719.
  Privé, G.G. 2007. Detergents for the stabilization and crystallization of membrane proteins. Methods 41:388‐397.
  Sellick, C.A., Hansen, R., Jarvis, R.M., Maqsood, A.R., Stephens, G.M., Dickson, A.J., and Goodacre, R. 2010. Rapid monitoring of recombinant antibody production by mammalian cell cultures using Fourier transform infrared spectroscopy and chemometrics. Biotechnol. Bioeng. 106:432‐442.
  Strop, P. and Brunger, A.T. 2005. Refractive index‐based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14:2207‐2211.
  Strug, I., Gutierrez, S., Cappione, A., Jimenez, M., Mullen, M.J., and Nadler, T. 2013. A rapid FT‐IR‐based method for monitoring detergent removal from biological samples. Spectroscopy 28:2‐6.
  Strug, I., Utzat, C., Cappione, A., Gutierrez, S., Amara, R., Lento, J., Capito, F., Skudas, R., Chernokalskaya, E., and Nadler, T. 2014. Development of a univariate membrane‐based mid‐infrared method for protein quantitation and total lipid content analysis of biological samples. J. Anal. Methods Chem. 2014, Article ID 657079. doi: 10.1155/2014/657079
  Tan, Y.J. and Ting, A.E. 2000. Non‐ionic detergent affects the conformation of a functionally active mutant of Bcl‐XL. Protein Eng. 13:887‐892.
Internet Resources
  https://www.applichem.com/en/literature/brochures/detergents
  Applichem, 2008. Detergents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library