Effective Removal of Nonionic Detergents in Protein Mass Spectrometry, Hydrogen/Deuterium Exchange,...

Effective Removal of Nonionic Detergents inProtein Mass Spectrometry, Hydrogen/DeuteriumExchange, and Proteomics

Martial Rey,† Hynek Mrazek,‡,§ Petr Pompach,‡,§ Petr Novak,‡,§ Ludovic Pelosi,†

Gerard Brandolin,† Eric Forest,| Vladimır Havlıcek,‡,⊥ and Petr Man*,‡,§,|

Laboratoire de Biochimie et Biophysique des Systemes Integres (BBSI), Institut de Recherches en Technologies etSciences pour le Vivant (iRTSV), UMR 5092 CNRS-CEA-UJF, Grenoble, F-38054, France, Laboratory of MolecularStructure Characterization, Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Vıdenska1083, Prague 4, CZ-142 20, Czech Republic, Department of Biochemistry, Faculty of Science, Charles University,Hlavova 8, Prague 2, CZ-12840, Czech Republic, Laboratoire de Spectrometrie de Masse des Proteines, Institut deBiologie Structurale, UMR 5075CNRS/CEA/UJF, 41 rue Jules Horowitz, Grenoble, F- 38027, France, and Departmentof Analytical Chemistry, Faculty of Science, Palacky University, Tr. Svobody 8, Olomouc, CZ-77146, Czech Republic

Detergents are frequently used for protein isolation andsolubilization. Their presence is crucial in membraneprotein protocols or in lipid raft proteomics. However,they are usually poorly compatible with mass spectrom-etry. Several different sample preparation protocols areroutinely used, but they are either laborious or suffer fromsample losses. Here, we describe our alternative methodfor nonionic detergent removal. It is based on selectivedetergent extraction after capture of the sample on areversed phase cartridge. The extraction is performed bychlorinated solvents and works well for polyoxyethylenebased nonionic detergents, but also for polymers likepolyethylene and propylene glycol. Detergent removal canbe also carried out on the protein level but a special caremust be taken with hydrophobic proteins. In such cases,it is preferable to perform detergent removal after pro-teolysis which digests the protein to peptides and reducesthe hydrophobicity. The method can easily be automatedand is compatible with hydrogen/deuterium exchangecoupled to mass spectrometry.

Detergents represent indispensible tools in protein research.They are widely used for isolation and solubilization of membraneproteins, isolation of lipid rafts, or as antiaggregating agents inprotein in vitro folding and crystallization.1-4 Even though theycan prove extremely useful, their presence may also havetremendous impact on the protein sample or its subsequentanalysis by mass spectrometry (MS). MS is a well established

technique for protein identification, characterization of its primarystructure, including modifications, and it has also found its placein the field of structural biology. Protein chemical modifications,cross-linking, hydrogen/deuterium (H/D) exchange, native elec-trospray ionization, and ion-mobility MS are examples of MS basedtechniques that provide structural biologists with invaluableinformation.5-9 Unfortunately, MS is often poorly compatible withthe presence of ionic detergents and has limited tolerance for thenonionic detergents (NID). Depending on the concentration,detergents may cause signal suppression, adduct formation, andshape distortion of protein signals.10-12 Polyoxyethylene baseddetergents (POE) negatively affect the analysis even at very lowconcentrations.13,14 Alkyl-glycosides are less harmful and reason-able spectra can be acquired in their presence, although they maycause charge state shifts in the protein spectra.10 Interestingly, arecent report described their utility in the analysis of intactmembrane protein complexes by nanoelectrospray ionization (ESI)MS.15 It was also shown that submillimolar concentration of alkyl-

* Corresponding author: (phone) +420-241062631, (fax) +420-241062156,(e-mail) [email protected].

† Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV),UMR 5092 CNRS-CEA-UJF.

‡ Academy of Sciences of the Czech Republic.§ Charles University.| Institut de Biologie Structurale, UMR 5075CNRS/CEA/UJF.⊥ Palacky University.

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10.1021/ac100171m 2010 American Chemical Society 5107Analytical Chemistry, Vol. 82, No. 12, June 15, 2010Published on Web 05/27/2010

glycosides enhances MALDI ionization of hydrophobic proteinsand peptides.16,17

Studies searching for the minimal tolerable amounts of variousdetergents have been conducted in order to discover how to handleMS applications where usage of detergents is necessary.10,11,17,18

Overall, matrix assisted laser desorption-ionization (MALDI) wasshown to be less sensitive to the presence of detergents than ESI.Detergents compatible with MS and cleavable detergents havealso been described.19,20 Acid-labile surfactants (ALS) are the mostwidely used type of cleavable detergents.21-23 Upon cleavage, theinsoluble hydrophobic part of ALS is removed by centrifugationwhile the soluble (hydrophilic) part, noninterfering with MSanalysis, remains in solution. Yu et al. has shown that loss ofhydrophobic peptides due to their separation into the precipitatedhydrophobic product occurs during the detergent cleavage/precipitation, which often can result in the loss of importantinformation about the analyzed protein.24

Another option is to develop different protocols for extractionof membrane proteins that avoid usage of detergents. Theseprotocols are based on the use of various organic solvents, acids,and their mixtures.25-27

Despite the improvements listed above there are many situa-tions where traditional detergents have to be used as thealternative procedures are not applicable. In such cases, detergentremoval methods are of high interest and so far many protocolsfor detergent removal have been described in the literature. Ingeneral, we can roughly divide them up into two groups: methodsremoving the detergent on the protein level and on the peptidelevel. In addition, methods can be classified according to whetherthey use native or non-native procedures. Native methods arealways conducted on the protein level. They encompass dialysis,gel filtration,28 adsorption on polystyrene beads,29 or trapping intocyclodextrin.30 Unfortunately, native techniques are only suitablefor detergent exchange or for decreasing the detergent concentra-tion because the complete removal (or decrease of detergentconcentration below critical micelle concentration) may lead toprotein precipitation. Non-native techniques employed at theprotein level are based on precipitation by acetone, trichloroacetic

acid, or other organic based solvent.31,32 The use of polyacrylamidegels for trapping of the protein and detergent removal was alsoreported.33 Finally, solvent extraction, chromatographic methods,or specific precipitation protocols are aimed at the specificphysicochemical properties of a detergent.25,34-36

Removal of detergents at the peptide level (e.g., after proteoly-sis) is mainly based on two principles, chromatography orextraction. Chromatographic methods separate the peptides fromdetergent based on their different hydrophobicity or charge. Ionexchange chromatography works well for ionic detergents37 butcan be also used for selective trapping of peptides from mixtureswith nonionic detergents. Specific arrangements include mixedbed resin (e.g., NID trap from MichromBioresources). Separationbased on hydrophobicity utilizes HILIC columns from which theNID elutes first and the peptides are eluted by decreasingpercentage of organic solvent. Despite several advantages, HILICcolumns do not offer separation efficiency comparable to reversedphase (RP) chromatography. Yeung et al. have used ethyl acetatefor quantitative removal of alkyl-glycosides and for partial removalof POE-based detergents like Triton X-100 and NP-40.38 Anotherwork, also based on ethyl acetate extraction, described removalof ionic sodium desoxycholate.39 Extraction protocols similar tothose mentioned above for intact proteins can be applied to peptidemixtures as well. One advantage is that due to the digestion largehydrophobic polypeptides are very often split into shorter and lesshydrophobic peptides.

Nevertheless, all techniques for detergent removal are suffer-ing from some drawbacks. As stated above, dialysis and adsorptiontechniques are more suitable for detergent exchange or forlowering their concentration. Precipitation can be problematic withsmall sample amounts and some proteins may be difficult toredissolve. The use of polyacrylamide gels may result in lowerrecovery or loss of larger and hydrophobic peptides. Specificprecipitations may cause losses of hydrophobic protein/peptides,as these can coprecipitate with the detergent. Chromatographictechniques can, especially in the ion exchange mode, also sufferfrom losses of specific peptides. This behavior depends on theconditions of chromatographic separation and represents aproblem mainly for complicated peptide mixtures covering a broadrange of isoelectric points. In the HILIC mode, phase separationmay occur at higher salt concentrations. Extraction with organicsolvents is not very efficient and several extraction steps must beperformed to remove the detergent quantitatively. In addition,hydrophobic peptides can easily be lost as they may partition intothe organic phase and, with lower volumes, it might be difficultto aspirate the solvent from the organic phase due to low viscosityand convex meniscus. But the major disadvantage of extraction

(15) Barrera, N. P.; Di Bartolo, N.; Booth, P. J.; Robinson, C. V. Science 2008,321, 243–246.

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2000, 72, 1169–1174.(18) Zhang, N.; Li, L. Rapid Commun. Mass Spectrom. 2004, 18, 889–896.(19) Ishihama, Y.; Katayama, H.; Asakawa, N. Anal. Biochem. 2000, 287, 45–

54.(20) Loo, R. R.; Loo, J. A. Anal. Chem. 2007, 79, 1115–1125.(21) Jaeger, D. A.; Jamrozik, J.; Golich, T. G.; Clennan, M. W.; Mohebalian, J.

J. Am. Chem. Soc. 1989, 111, 3001–3006.(22) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Anal. Chem.

2003, 75, 6023–6028.(23) Chen, E. I.; Cociorva, D.; Norris, J. L.; Yates, J. R. J. Proteome Res. 2007,

6, 2529–2538.(24) Yu, Y. Q.; Gilar, M.; Gebler, J. C. Rapid Commun. Mass Spectrom. 2004,

18, 711–715.(25) Barnidge, D. R.; Dratz, E. A.; Jesaitis, A. J.; Sunner, J. Anal. Biochem. 1999,

269, 1–9.(26) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.;

Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351–360.(27) Hjelmeland, L. M. Methods Enzymol. 1990, 182, 253–264.(28) Hjelmeland, L. M. Methods Enzymol. 1990, 182, 277–282.(29) Rigaud, J. L.; Levy, D.; Mosser, G.; Lambert, O. Eur. Biophys. J. 1998, 27,

305–319.(30) DeGrip, W. J.; VanOostrum, J.; Bovee-Geurts, P. H. M. Biochem. J. 1998,

330, 667–674.

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56, 734–749.(35) Shively, J. E. In Methods of Protein Microcharacterization; Shively, J. E.,

Ed.; Humana Press: Clifton, 1986; pp 41-87.(36) Zhou, J.; Zhou, T.; Cao, R.; Liu, Z.; Shen, J.; Chen, P.; Wang, X.; Liang, S.

J. Proteome Res. 2006, 5, 2547–2553.(37) Vissers, J. P.; Hulst, W. P.; Chervet, J. P.; Snijders, H. M.; Cramers, C. A.

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techniques remains in their “offline” arrangement. They cannot,or only with some limitations,40 be applied to online LC-MSanalysis, for instance, for MS based H/D exchange.

Here, we describe a new method for removal of NIDs basedon a combined principle of chromatographic separation andorganic solvent extraction. This approach was tested in an onlinecoupling of liquid chromatography (LC) and MS, for hydrogen-deuterium amide exchange coupled to mass spectrometry andwe also applied the technique to analysis of hydrophobic proteins/peptides.

EXPERIMENTAL SECTIONChemicals. All chemicals were from Sigma Aldrich unless

otherwise stated. Solvents were from Merck. Isolation of bovineADP/ATP carrier was done according to the protocol publishedpreviously with minor modifications.41 Cyclosporin A and Efrapep-tin were generous gifts of Dr. Alexandr Jegorov from Teva CzechIndustries, Czech Republic.

Sample Preparation. Bovine serum albumin was digestedovernight in 50 mM ammonium carbonate buffer pH 8.0 withtrypsin (w/w ratio 1:50) at 37 °C. Following digestion, the solutionwas acidified with formic acid (FA), aliquoted (1 pmol/tube), andstored frozen. Bacteriorhodopsin was dissolved in 70% trifluoro-acetic acid (TFA) and cyanogen bromide (CNBr) was added to afinal concentration of 20 mg/mL. Digestion was carried outovernight under argon. Next day, the solution was aliquoted (5pmol/tube) and dried in a SpeedVac concentrator. Prior to theexperiment, the aliquot was dissolved by stepwise addition offormic acid, acetonitrile (ACN), and water so that the finalconcentration of ACN and FA was 5% and 0.4%, respectively. Apeptide Microtrap in the off-line holder (MichromBioresources,Auburn, CA) was used for the testing and off-line desalting/detergent removal. For testing of the different resins, homemadecolumns were created from Millipore Gel-loader tips.42 Solventsused for off-line desalting were water/0.4% FA (FA0), dichloro-or trichloromethane/0.4% FA (DCMFA or TCMFA), 45% ACN/54.6% H2O/0.4% FA (FA45), and 85% ACN/14.6% H2O/0.4% FA(FA85). The trap was cleaned between the experiments with80% ACN/10% isopropyl alcohol/9% H2O/1% FA.

Fully deuterated apomyoglobin was prepared by dissolutionof apomyoglobin in D2O followed by overnight incubation andconcentration on a Speed-Vac concentrator. The process ofdilution and concentration was repeated three times.

H/D exchange was initiated by a 10-fold dilution of the proteinsample into deuterated buffer containing 10 mM MOPS-NaOH,10 mM NaCl, 1 mM EDTA, pD 6.8 and performed at 4 °C.Exchange was quenched by 1 M Glycine-HCl (pH 2.5) and rapidfreezing in liquid nitrogen.

Liquid Chromatography. High-performance liquid chroma-tography was performed on Agilent HPLC 1100 series system(Agilent technologies, Santa Clara, CA) composed of an autosam-pler, capillary and nano pump both operating in the normal flowmode. The capillary pump was delivering solvents A and B at a

flow rate of 50 µL/min. Solvent A was 0.03% TFA in water and Bwas 95% ACN/5% H2O water/0.03% TFA (both pH 2.3). The nanopump was pumping solvents A and C where A was same as onthe capillary pump and C was chloroform/0.03% TFA. Valveswere from Rheodyne (Oak Harbor, WA), the trap cartridgefrom Michrom Bioresources and the column (Jupiter C18, 1 ×50 mm) from Phenomenex (Torrance, CA). The pepsin columnwas prepared according to the standard protocol.43 The entireHPLC setup (tubing, valves, trap column, and analyticalcolumn) was immersed in an ice-bath (0 °C) and the outlet ofthe column was directly interfaced to an electrospray ion sourceof a mass spectrometer.

Mass Spectrometry. Matrix-assisted laser desorption ioni-zation-time-of-flight (MALDI-TOF) mass spectrometry was doneon Ultraflex III (Bruker Daltonics, Bremen, Germany). Thesamples were directly mixed on the target with R-cyano-4-hydroxycinnamic acid and dried at room temperature. Spectrawere acquired in reflectron mode. Direct infusion-electrosprayionization (ESI) mass spectrometry analyses were performed ona Fourier transform ion cyclotron resonance (FT-ICR) massspectrometer (APEX-Qe, Bruker Daltonics, Bremen, Gremany)equipped with 9.4T superconducting magnet. Liquid chromato-graphy-mass spectrometry (accurate mass measurements andthe analysis of deuterated samples) was carried out on an ESI-TOF instrument (Agilent 6210, Agilent technologies, Santa Clara,CA). LC-MS/MS analyses were performed on an ion trap massspectrometer (Esquire 3000+, Bruker Daltonics, Bremen, Ger-many). MS/MS data were processed using MASCOT (MatrixScience, London, U.K.) and DrawMap script.44 Data mining ofthe protein sequences was done in GPMAW v. 7.1 (LighthouseData, Odense, Denmark).

RESULTS AND DISCUSSIONDevelopment of Detergent Removal Protocol. Protein mass

spectrometry and proteomics protocols often require detergentremoval prior to MS analysis. Because the traditional methodsare having some drawbacks and cannot be automated or easilyused for H/D exchange experiments, we actively searched for adifferent way of detergent removal. First we tested a protocol thatis based on extraction with organic solvents (dichloromethane,DCM, or Ethyl acetate, EA). Experiments were done with BSAtryptic digest (1 pmol; peptides found in tryptic BSA digest arelisted in Table S-1) spiked with 1% Triton X-100. Individualextraction steps were monitored by MALDI-TOF MS and theresults showed that several steps were required for a completeremoval. No loss of peptides was observed when the extractionwas done on a pure BSA digest, but when this protocol was testedon a more hydrophobic sample, a CNBr digest of bacteriorho-dopsin (analyzed by ESI-FT-ICR MS, list of detected peptides canbe found in Table S-2), partial loss of the most hydrophobicpeptides (e.g., 82-131 and 177-222) occurred. Peptide loss waseven higher (nearly complete) with very hydrophobic samplesrepresented by cyclic peptides Cyclosporin A and Efrapeptin. Thisis not surprising because extraction with chlorinated solvents or(40) Boersema, P. J.; Divecha, N.; Heck, A. J.; Mohammed, S. J. Proteome Res.

2007, 6, 937–946.(41) Brandolin, G.; Doussiere, J.; Gulik, A.; Gulik-Krzywicki, T.; Lauquin, G. J.;

Vignais, P. V. Biochim. Biophys. Acta 1980, 592, 592–614.(42) Thingholm, T. E.; Jorgensen, T. J.; Jensen, O. N.; Larsen, M. R. Nat. Protoc.

2006, 1, 1929–1935.

(43) Rey, M.; Man, P.; Brandolin, G.; Forest, E.; Pelosi, L. Rapid Commun. MassSpectrom. 2009, 23, 3431–3438.

(44) MSTools Web page. http://ms.biomed.cas.cz/MSTools/.

5109Analytical Chemistry, Vol. 82, No. 12, June 15, 2010

their mixtures is routinely used for isolation of these compounds.45,46

To overcome the loss of hydrophobic peptides, a modification ofthe extraction protocol was introduced.

This modification is based on the use of reversed phasecartridge as the vessel for extraction. Under such conditions,peptides are bound to the resin and this significantly lowers theirtendency to partition into the organic solvent phase. The solventwhich is used for detergent removal must fulfill two followingrequirements: low miscibility with water and high ability todissolve and thus extract the detergent.

BSA digest (1 pmol) spiked with 1% Triton X-100 was loadedon the peptide microtrap equilibrated in FA0. The sample wasdesalted with FA0, and in the next step, the detergent wasremoved by DCM. Following detergent removal, the peptides wereeluted with FA45. MALDI-MS of individual fractions showed thatthe peptides and detergent were efficiently trapped and no lossoccurred during the sample loading and desalting. A wash withDCM removed the detergent but still some remained boundbecause it eluted together with the peptides in the FA45 elutionstep. This was probably due to the fact that a small portion(corresponding to the trap dead volume) of DCM was releasedby the elution with FA45.

To evaluate the extent of possible peptide losses during thedetergent removal step, an experiment with pure BSA trypticdigest was performed. There was no peptide loss detected duringDCM elution. Further steps were aimed to the removal ofdetergent/DCM traces prior to FA45 elution. An additional washwas inserted between detergent removal and peptide elution.Different solvents, both miscible and immiscible with water(ethylacetate, acetone, etc.), were tested, but none of themprovided the expected result. Surprisingly, the simple wash withFA0 was able to remove all the DCM/detergent traces. This wasvisible on the collected fractions that were clearly showing twodifferent phases. The successful removal was confirmed by MSanalysis, where no detergent was detected in the FA45 fraction.However, selective loss of short, highly hydrophilic peptides wasobserved (e.g., 5-10 and 212-217, Table S-1). This was causedby the absence of ion pairing agent in the DCM wash. When theacidified DCM was used, the short hydrophobic peptides werenot lost and were detected in the FA45 fraction.

Other organic solvents, for example, EA and chloroform(TCM), were also tested for their ability to remove detergent.While TCM showed the same results as DCM, EA led (due to itshigher miscibility with water) to the loss of peptides in secondFA0 wash. Figure 1 shows an example of MALDI-TOF spectra ofall fractions from the experiment where DCMFA was used andits traces were removed by FA0. The advantage of this setup isthat the system is fully equilibrated prior to the gradient elution,and thus, the separation is not affected. This makes the methodsuitable for online connections.

Keeping in mind that the online connections are done withthe ESI sources and the ESI is much more sensitive to thedetergent than the MALDI, the detergent removal was alsoevaluated using ESI. Employing the setup described above, nodetergent traces were found in the peptide elution (FA45) but

small detergent amount was detected in the subsequent wash (80%ACN/10% isopropyl alcohol/9% H2O/1% FA). Stepwise elutionby increasing acetonitrile concentration (10% steps from 10 to90%) was tested on the sample after detergent removal. Nodetergent traces were observed up to 50%. Above 50%, somedetergent signal was found. However, it was lowered to a levelallowing detection of the peptides. In parallel with this experi-ment, direct stepwise elution of the BSA tryptic digest spikedwith Triton X-100 was also performed. Here, the detergentappeared already between 20 and 30% with majority elutingbetween 40% and 50% of ACN. Additional experiments alsoshowed that the detergent concentration detectable in MALDIis above 0.0001% and in ESI this level is even lower - 0.00001%.This means that our protocol does not remove the detergentcompletely but it lowers the detergent amount bellow thedetection limit of MALDI and to a level allowing the analysisusing ESI. To show that and evaluate the usefulness of themethod for the removal of other detergents, the method wasfurther tested on different POE detergents (namely, Brij 58,Brij 96, Brij 98, NP-40, Tween-80, Triton X-114) and on polymerslike polypropylene (PPG-2700) and polyethylene (PEG-4000)glycol. In all cases, the detergent/polymer was nearly com-pletely removed from the peptides as shown on examples ofBrij-58, PEG-4000, Triton X-100 and Tween-20 (Figure 2, ESI;and Figure S-2, MALDI). Small variations in the efficiency of thedetergent removal were found between DCMFA and TCMFA withTCMFA being more suitable for all tested compounds (betterefficiency with Brijs). Interestingly, the same signal for POE basedcompound was detected in all samples, indicating the contamina-tion introduced by chlorinated solvents. Its intensity is rather lowand can be nearly diminished if high-quality DCM or TCM isredistilled in the laboratory and used fresh.

Further experiments were focused chiefly on the hydrophobicpeptides. CNBr digested bacteriorhodopsin was again chosen asa model sample. Even the most hydrophobic peptides weredetected in elution with FA85 (here the percentage of organicsolvent was raised to 85% due to higher hydrophobicity of theanalyte) and no loss was observed. Unfortunately, the sameprotocol could not be used for cyclic peptides because they werealmost completely eluted by TCMFA or DCMFA and otherdifferent resins as stationary phases need to be tested.

The Influence of Stationary Phase on Detergent Removal.The peptide trap originally used in our experiments is packed withpolymeric beads with retentivity similar to C8 or phenyl resins.To gain a better understanding of the influence of the resin andits chemical properties on the separation, several other resins weretested and compared using offline detergent removal coupled toMALDI-MS. The resins tested included trap columns (Protein(polymeric ∼ C4) and Small molecule trap (silica based C18) fromMichrom Bioresources), commercially available pipet tips (Mil-lipore, ZipTip C18; Varian, OMIX C18; Agilent, Peptide Cleanuptips C18) or lab-made packed pipet gel loader tips (AppliedBiosystems, R1 and R2 POROS resins; Michrom Bioresources,silica based MAGIC C4, C8, C18 and C30). Unfortunately, wefound that some of the commercial pipet tips are not suitable forour method because the packing is washed away when DCMFAor TCMFA is applied. The lab-made tips may suffer from carryoverand from insufficient detergent removal both caused probably by

(45) Kuzma, M.; Sedmera, P.; Jegorov, A.; Havlicek, V. J. Nat. Prod. 2009, 72,159–163.

(46) Wu, J.; Chatman, K.; Harris, K.; Siuzdak, G. Anal. Chem. 1997, 69, 3767–3771.


the inhomogeneity of packing. Short alkyl chains or resins withlow hydrophobicity (C4 or Protein Trap) exhibited peptide lossduring loading, detergent removal, and second FA0 wash. On theother hand, with long alkyl chains, we found traces of detergentin the FA45 wash. Closer inspection of the spectra showed thatthe detergent eluted in FA45 wash does not represent thecomplete envelope covering the whole distribution of polyoxy-ethylene chain lengths but the spectra show only the species withlonger chains (less hydrophobic ones). In addition, the other partof the envelope covering the shorter chains representing morehydrophobic part of the detergent eluted in TCMFA or DCMFAwash. Since C18 and C30 resins were only silica based, we canaccount for this phenomenon by the interaction with free silanoylgroups. Regardless of this observation, we turned back to ourinitial idea that the more hydrophobic resins can be suitable fordetergent removal from cyclic peptides. Both of the hydrophobic

cyclic peptides, Efrapeptin and Cyclosporin, exhibited only a smallloss during TCMFA or DCMFA wash. They were eluted withFA85 together with the detergent portion covering the lesshydrophobic part of the detergent. However, the concentrationof the detergent was lowered to a level compatible with electro-spray ionization because the signal for cyclic peptides wasdetected. This is in contrast to the initial solution where onlydetergent signal was detected and no cyclopeptides were recorded.It can be concluded that the method is not suitable for detergentremoval from extremely hydrophobic peptides as they are notretained by polymeric ∼ C8 resin and more hydrophobic, silicabased C18 or C30 retains a significant portion of the detergent.With respect to the observation that the detergent is not removedabsolutely, the effect of porosity was also evaluated. NonporousRP resin was packed into a guard column cartridge and thedetergent removal was performed on BSA tryptic digest spiked

Figure 1. Individual steps of detergent removal on polymeric peptide MicroTrap monitored by MALDI-TOF MS. (A) 1 pmol of BSA tryptic digestspiked with 1% Triton X-100 before loading onto the trap; (B) flow-through fraction and desalting with FA0; (C) detergent removal by DCMFA:(D) removal of DCM traces and re-equilibration with FA0; (E) peptide elution with FA45; (F) wash of the peptide trap with 80% ACN/10%isopropyl alcohol/9% H2O/1% FA. Asterisk denotes matrix adduct peaks.


with different detergents. Likewise, in the case of the peptide trap,some detergent traces were observed in ESI. Therefore, thetrapping of the remaining detergent traces is not influenced bythe porosity of the resin.

Applications in Proteomics. After the optimization of deter-gent removal, we turned our attention to specific applications ofthe protocol. In all subsequent parts, the Peptide trap (polymeric∼ C8) was used. Solvent for detergent removal was TCMFA andtrap re-equilibration was accomplished by acidified water (by FA0or its alternative with trifluoroacetic acid instead of formic acid).

Many protocols in protein mass spectrometry (sample process-ing after immunoprecipitation or isolation of membrane micro-domains) employ acetone or trichloroacetic acid precipitation fordetergent removal. As mentioned above, these protocols are notfully reproducible with small sample amounts and suffer fromsample losses or unwanted modifications (e.g., formylation,

esterification, carbamylation, etc.) introduced during redissolutionunder harsh conditions. In the modified procedure, the protocolstarts with digestion followed by the detergent removal accordingto the protocol described in the previous paragraphs. We arecurrently using this method for preparation of digests from lipidrafts. Preliminary results show that our protocol provides betterreproducibility than the precipitation. Here, we show the com-parison of various protocols for detergent removal on BSA (Table1 and Figure S-1). In this experiment, various amounts of BSAwere spiked with Triton X-100 and processed either by gelelectrophoresis with in-gel digestion, by TCA precipitation, or byour protocol. The data in the Table 1 and spectra in Figure S-1clearly show that the precipitation led to the extensive losseswhereas our protocol for detergent removal outperformed eventhe gel electrophoresis.

Figure 2. Removal of different detergents monitored by ESI-MS (direct infusion). BSA tryptic digest spiked with Brij-58, Tween-20, TritonX-100, or PEG-4000 was subjected to offline manual detergent/polymer removal on a peptide trap (DCMFA wash followed by FA0 wash andelution with FA85). Spectra of the initial solutions are shown in column A. Spectra of samples after detergent removal are listed in column B.The third column (C) shows the data deconvoluted from the spectra in column A (in gray) and B (in black). Complete removal of the contaminantswas achieved for Tween-20 and PEG-4000 and nearly quantitative removal was achieved for Triton X-100 and Brij-58. From the deconvolutedspectra, it is also evident that some common POE based compound (m/z 509, 553, 597, 641, 685, 729, 773, 817, 861) appears in all thesamples.


We also show that the protocol can be applied to intact proteinsas well. Here, care must be taken with membrane and highlyhydrophobic proteins. However, smaller or less hydrophobicproteins can be also separated from the detergent. Figure S-3shows the detergent removal on a recombinant protein, extracel-lular part of rat NK cell receptor NKR-P1A (provided by O. Vanek,Institute of Microbiology in Prague).

Evaluation of Online Detergent Removal in Liquid Chro-matography and Its Compatibility with H/D Exchange. Firststep on the way to the online applications was the evaluation ofLC separation with and without detergent removal. BSA trypticdigest was injected either alone or spiked with a mixture ofdetergents (Triton X-100, Brij-58, Tween-20) and polymer (PEG-4000) onto the LC-MS system composed of the LC pumps, trapcartridge, analytical column and ESI-qIT. After the sample injec-tion, the trap cartridge was desalted, disconnected from thesystem, and offline washed with TCMFA. After that, it was placedback and TCMFA was removed. Next, the gradient elution 10-50%of solvent B in 30 min, 50% solvent B in 10 min, and 50%-90%

solvent B in 10 min was done. The example chromatogramsshowing separation of pure BSA tryptic digest (Figure S-4A) andthe digest spiked with detergent mixture (Figure S-4C), both afterdetergent removal, are shown. The trace corresponding to theseparation of detergent spiked digest on which the detergentremoval was not performed (cartridge was not washed withTCMFA) is also shown (Figure S-4B). It is evident that withoutthe removal the chromatogram is dominated by the peakscorresponding to the separation of the polymeric molecules asdescribed also by others.14 The example spectra (insets) also showthat in the presence of the detergent the intensity of the peptidepeaks is strongly reduced especially for peptides with m/zoverlapping with the detergent signals. The remaining traces ofthe detergent after detergent removal are not separated but eluteas a small peak in the second part of the chromatogram.

Evaluation of the Back-Exchange in H/D Experiments.The next set of experiments was focused on the applicability tohydrogen/deuterium exchange monitored by mass spectrometry.In this method, the speed of sample processing is crucial and therestrictions given by the principle of H/D exchange must befulfilled in order to minimize experimental error caused byback-exchange.47,48 It is also beyond any doubt that the possibilityof automation speeds-up the workflow and improves reproduc-ibility. A chromatographic system allowing online protein diges-tion, desalting, and detergent removal prior to chromatographicseparation (Figure 3) was set up, placed in an ice-bath, andconnected to an ESI-TOF instrument. Fully deuterated recombi-nant sperm-whale apomyoglobin was selected as a model example.Deuteration levels of selected peptides, previously used in a studyon aMb interaction with lipidic vesicles,7 were monitored. Com-parison of back-exchange was done in three experiments differingin the sample processing. The first experiment encompassedsimple online digestion and desalting. The sample was injectedthrough the flow path of the pump P1 (Figure 3) onto the pepsincolumn and the resulting peptides were trapped on a peptidemicrotrap (Figure 3A). After the digestion and desalting for 1.5min by solvent A, valve 2 was switched and gradient elutionstarted (Figure 3C). The second experiment comprised additionalsteps for detergent removal and trap re-equilibration. Afterdigestion and desalting (1.5 min), the valve 1 was switched andsolvent C was pumped through the trap for 1 min for detergentremoval (Figure 3B). Next, the trap was re-equilibrated for 1.5min with solvent A. Following digestion, desalting, and detergentremoval, both valves, 1 and 2, were switched and gradient elutionof the peptides from trap onto the analytical column was done(Figure 3C). The last experimental setup was the same as theprevious one with only one exception that the wash with solventC was replaced by wash with solvent A. In all cases, the separationof peptides was done by a gradient elution (pump P2) from 10%B to 50% B within 20 min. Extracted ion chromatograms ofselected peptides were plotted, signals were averaged, and thespectra exported and processed by MagTran.49 Deuterationpercentage was calculated as a measured mass of a peptide dividedby the theoretical mass of a fully deuterated peptide. The resultsof triplicate measurements are shown in Figure 4.

(47) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522–531.(48) Wales, T. E.; Fadgen, K. E.; Gerhardt, G. C.; Engen, J. R. Anal. Chem. 2008,

80, 6815–6820.(49) Zhang, Z.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 225–233.

Table 1. BSA Tryptic Peptides Found in the Digestfrom Samples Containing Triton X-100

10 µg 1 µg 0.1 µg

M+H+ From-To A B C A B C A B C

609.288 500-504 1 0 1 1 0 1 0 0 0649.334 181-185 1 1 1 1 0 1 0 0 0689.373 212-217 1 1 1 1 1 1 0 0 1712.374 5-10 1 1 1 1 0 1 1 0 1841.460 459-465 1 0 1 0 0 1 0 0 0927.493 137-143 1 1 1 1 1 1 1 1 1974.458 13-20 1 1 1 1 0 1 0 0 11022.439 475-483 1 1 1 0 0 1 0 0 11050.493 564-573 1 1 1 0 0 1 0 0 11163.631 42-51 1 1 1 1 1 1 1 1 11249.621 11-20 1 1 1 1 1 1 1 1 11283.711 337-347 1 1 1 1 1 1 1 1 11305.716 378-388 1 1 1 1 1 1 1 0 11347.530 52-64 1 1 1 1 0 1 0 0 11362.672 65-76 1 0 1 1 0 1 0 0 01399.693 545-556 0 0 1 0 0 1 0 0 11439.812 336-347 1 1 1 1 1 1 1 1 11479.795 397-409 1 1 1 1 1 1 1 1 11511.843 414-427 1 1 1 1 1 1 0 1 11567.743 323-335 1 1 1 1 1 1 1 1 11639.938 413-427 1 1 1 1 1 1 1 1 11667.813 445-458 1 1 1 0 0 1 0 0 11823.900 484-499 1 1 1 1 1 1 0 1 11888.927 145-159 1 1 1 1 1 1 0 0 12045.028 144-159 1 1 1 1 1 1 1 1 12222.890 199-204 +

243-2561 1 1 0 0 1 0 0 1

Sequence coverage(in %)

39 35 42 31 22 42 19 19 37

a Three different amounts (10, 1, and 0.1 µg) of BSA were spikedwith 1% Triton X-100 and processed by three different protocols. Firstcolumn shows monoisotopic M+H+, second column shows the peptidelimits. Presence (1, in bold) or absence (0, in italics) of individualpeptides is shown in the following columns for all conditions and BSAamounts. Conditions: A, gel electrophoresis followed by in-gel digestionand desalting; B, trichloroacetic acid precipitation, washing of the pelletwith acetone, chloroform/methanol and ethanol/ether followed by re-dissolution, in-solution digestion and desalting; C, direct digestion insolution followed by desalting/detergent removal. In all cases, thepeptides were in the last step of cleanup eluted with 50 µL of FA45and 1µL was deposited on the MALDI target. Final amount of thesample on the target was therefore 3 pmol, 300 fmol, and 30 fmol,respectively. Sequence coverage for individual conditions is shown atthe bottom of the table.


It is evident that detergent removal was not causing significantback-exchange. The average deuterium recovery was only by 1.3%lower than the one obtained after classical short desalting. Therefore,we can conclude that the detergent removal by acidified chlorinatedsolvents is not causing significant back-exchange and the methodcan be applied to H/D exchange monitored by mass spectrometry.

Application of the Protocol to H/D of Membrane Proteins.In the last experiment, the method was tested on a membraneprotein, ADP/ATP carrier isoform 1 (bAnc1p), isolated frombovine heart mitochondria. The protein in buffer containing

approximately 10% of Triton X-100 (the exact concentration is notknown due to concentration steps in the sample preparation) wasinjected onto the system described above and shown in Figure 3.The system was connected either to an ion trap operating inLC-MS/MS (peptide identification) mode or to an ESI-TOFinstrument (verification of the MS/MS assignments based onaccurate mass and analysis of deuterated samples). As in previouscases, online digestion followed by desalting, detergent removal,and re-equilibration was performed under H/D compatible condi-tions (pH 2.3 and 0 °C). The peptides were separated by a gradientelution from 10% to 50% B using the same solvent system asdescribed in the previous paragraph. The MS/MS data weresearched by MASCOT and the assignments verified by accuratemass measurements. The results were processed by the DrawMapprogram. Peptides, including the modified ones (N-terminalacetylation of Ser and trimethylation of Lys51), covering 94% ofthe entire sequence, were successfully identified (Figure 5). Themissing parts are represented by very short stretches which werenot retained by the reversed phase resin or were probably digestedto di- or tripeptides. Figure 5 also shows the examples of base-peak chromatograms obtained after separation of the sample fromwhich the detergent was (Figure 5A, top) or was not removed(Figure 5A, bottom). As in the case of spiked BSA tryptic digest(Figure S-4B), even here (Figure 5A, bottom) the chromatogramshows separation of the polymeric molecules. The example spectra(Figure 5A, insets) again nicely demonstrate peptide signalincrease after detergent removal. Work focused on the H/Dexchange of this protein is currently in progress (Rey, M.; et al.,in preparation). Interestingly, the washing procedure removed not

Figure 3. Semiautomated HPLC setup for online digestion, desalting, detergent removal, and separation. (A) Connections during online digestion,peptide trapping, and desalting (pump P1); (B) detergent removal and re-equilibration (pump P1); (C) peptide trap elution/gradient separation(pump P2). Flow path of pump P1 is shown as a solid black line, flow path of pump P2 is shown as a dashed black line. Inactive flow path isin gray.

Figure 4. Deuterium recovery of aMb peptic peptides after differentsample processing. Black, desalting for 1.5 min with solvent A. White,desalting for 1.5 min followed by 1 min of detergent removal by solventC and 1.5 min of re-equilibration by solvent A. Gray, desalting for 4min by solvent A. The graph shows the results of triplicate measure-ment with standard deviation shown as error bars.


only the detergent but also native lipids, which were present inthe sample. It is also noteworthy that no differences in theefficiency of detergent removal were found when the separationof nondeuterated sample was done at the ambient temperature.

This shows the feasibility of the method in the field of MSanalysis of membrane proteins and especially in H/D exchange.Because highly hydrophobic peptides can be lost during thedetergent removal, we should point out that the type of the enzyme

Figure 5. Mapping of bovine ADP/ATP carrier 1. Protein in buffer with 10% Triton X-100 was injected onto the system shown in Figure 3.Digestion, desalting, and detergent removal were performed online before LC-MS/MS analysis on an ESI-qIT or LC-MS analysis on an ESI-TOF. Panel A shows the examples of base peak chromatograms (LC-MS) after detergent removal (top) and without detergent removal (bottom).Example ESI-TOF spectra (averaged over the region highlighted by a gray bar) where the N-terminal peptide (residues 1-7) was detected areshown as insets. The top inset also includes the isotopic pattern of the peptide 1-7 after H/D exchange. Panel B shows the sequence coverage(94%) based on the peptides identified in LC-MS/MS run. Gray bars bellow the sequence represent the identified peptides. Secondary structureelements (R-helices H1-H6 and interconnecting R-helices h12-h56) are shown as cylinders above the sequence.


chosen for the digestion can influence the recovery and sequencecoverage. In studies dealing with membrane proteins, specificproteases working at basic pH are often used. They facilitate dataprocessing and provide peptides with better ionization properties(two basic sites, N-terminus and Lys or Arg at the C-terminus, incase of trypsin), but unfortunately, they often have few cleavagesites in the sequence, especially in the transmembrane regions.As a result, large, hydrophobic peptides are obtained afterenzymatic digestion. One way to decrease the hydrophobicity isto cleave the protein using cyanogen bromide since the methion-ine occurrence in the transmembrane segments is much higherthan that of basic amino acids.50 The other is to utilize nonspecificacid proteases, which are routinely used in H/D exchange.Commonly used pepsin cleaves at the C-terminus of hydrophobicamino acids. Fungal acid proteases (protease type XIII or typeXVIII), splitting the peptide bond at the C-terminus of hydrophobicand basic amino acids, are also becoming very popular.43,51 In allcases, the digestion leads to generation of less hydrophobicpeptides, although rather complex mixtures are produced. This,however, can be dealt with thanks to currently available high-resolution mass spectrometers and modern dissociation tech-niques.52-55 Such instrumentation can easily handle problems withthe ragged cleavage, higher complexity of the sample, and absenceof basic sites at the C-terminus. That is why it seems advantageousto use nonspecific proteases for digestion of membrane proteinsalso in all proteomics protocols, not only in H/D exchange.

CONCLUSIONA new method for removal of polyoxyethylene based deter-

gents and similar compounds from samples containing peptides

or proteins was introduced. It is based on the binding of thedetergent/polymer containing mixture onto a reversed phase resinfollowed by a selective wash of the nonionic contaminants bychlorinated solvents. The usability for detergent removal wasdemonstrated mainly on the peptide level, but the techniqueproved to be useful on the protein level as well, although in thisarea it may be limited to soluble proteins. The method was testedthoroughly with different polymers/detergents, different reversedphases and with the solvents suitable for polymer/detergentremoval. It was shown that the detergent is not removedcompletely but its remaining level is bellow the sensitivity ofMALDI and just slightly above the threshold level of ESI. Themethod can be easily automated; the efficiency of detergentremoval is not affected by the temperature. The detergent removalprocedure is also compatible with hydrogen/deuterium exchangecoupled to mass spectrometry.

ACKNOWLEDGMENTThe first two authors contributed equally to this work. The

authors thank Michael Volny for helpful discussions and criticalreading of the manuscript and Martin Strohalm for help with thefigures. We also thank all the reviewers for their excellentcomments. Financial support from the Grant Agency of theAcademy of Sciences of the Czech Republic (KJB500200612);Institutional research concept of the Institute of Microbiology(AVOZ50200510), Ministry of Education, Youth and Sports of theCzech Republic (LC545), and funding from the UJF, the CentreNational de la Recherche Scientifique (CNRS) and the Commis-sariat a l’Energie Atomique (CEA) is gratefully acknowledged.Martial Rey is supported by a fellowship from the UniversiteJoseph Fourier (UJF).

SUPPORTING INFORMATION AVAILABLEAdditional information as noted in text. This material is

available free of charge via the Internet at http://pubs.acs.org.

Received for review January 20, 2010. Accepted May 6,2010.

AC100171M

(50) Eichacker, L. A.; Granvogl, B.; Mirus, O.; Muller, B. C.; Miess, C.; Schleiff,E. J. Biol. Chem. 2004, 279, 50915–50922.

(51) Zhang, H. M.; Kazazic, S.; Schaub, T. M.; Tipton, J. D.; Emmett, M. R.;Marshall, A. G. Anal. Chem. 2008, 80, 9034–9041.

(52) Kazazic, S.; Zhang, H. M.; Schaub, T. M.; Emmett, M. R.; Hendrickson,C. L.; Blakney, G. T.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2010,21, 550–558.

(53) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev.1998, 17, 1–35.

(54) Perry, R. H.; Cooks, R. G.; Noll, R. J. Mass Spectrom. Rev. 2008, 27, 661–699.

(55) Kaltashov, I. A.; Bobst, C. E.; Abzalimov, R. R. Anal. Chem. 2009, 81, 7892–7899.


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