created: 11th December 1997, last updated: 11th December 1997,© 1998 ABRF


Kristine M. Swiderek1, Andrew J. Alpert2, Amos Heckendorf3, Kerry Nugent4 & Scott D. Patterson5

1Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010; 2PolyLC Inc., 9151 Rumsey Rd., Suite 180, Columbia, MD 21045; 3The Nest Group, 45 Valley Rd., Southborough, MA 01772; 4 Michrom BioResources, 1945 Industrial Drive, Auburn, CA 95603, 5Amgen Inc., 1840 DeHavilland Dr., Thousand Oaks, CA 91320.


This article has been written as a review of some of the methods available to prepare detergent-containing protein and peptide samples prior to structural analysis techniques. We address issues such as the removal of ionic and nonionic detergents using a variety of techniques and offer strategies when, for various reasons, a detergent cannot be removed from the sample. Although this article does not cover all possible scenarios, we hope that it will at least serve as a starting point.


Detergents seem to be a necessary evil for the protein chemist. The successful preparation of different protein and peptide samples often relies on their presence. They have proven beneficial in a large number of routine laboratory techniques (1-6). However, the presence of detergents can also result in denaturation of proteins and can be detrimental to the activity of proteolytic enzymes. Detergents can also heavily interfere with purification procedures such as chromatography and gel electrophoresis as well as with techniques used for structural characterization such as mass spectrometry, amino acid and sequence analyses. Over the years, strategies for the use of detergents have been successfully refined to reduce negative effects on various analytical techniques. However, with the increased use of several new and more sensitive methods for protein analysis, such as mass spectrometry, the problems associated with detergents are again on the rise. The recently developed mass spectrometric-based protein identification strategies, peptide-mass searching (5, 7, 8) and tandem mass spectrometry (MS/MS) (9-11), require compatibility with electrophoresis, digestion, and elution protocols. Although detergents can interfere with this type of analysis, different approaches can be taken to overcome the obstacles created by them in protein and peptide samples: (A) removing or reducing the amount of detergent, (B) substituting interfering detergents with non-interfering detergents, (C) eliminating the use of detergents throughout the protocols, and/or (D) adjusting the analytical technique so that the presence of detergent does not conflict with the analysis. Throughout this article, we will try to explore each of these options with different examples.

General Detergent Properties

Detergents are surface-active agents (surfactants) containing a hydrophobic portion, which is more soluble in oil-like solutions, and a hydrophilic portion, which is soluble in water. Detergents will migrate to the interface of a solvent reducing its surface tension. At low concentration, detergents will form monolayers; at higher concentration, also called the critical micellar concentration (CMC), they tend to aggregate to form micelles or clusters. Detergents are distinguished by their physicochemical properties. These properties must be carefully considered when choosing a detergent for purification or analytical techniques. Table 1 gives an overview of the different types of detergent and some examples of each (12).

Choose this link to View Table 1 (it will take a little time to download)

Removal of Detergents

Classical Methods for the Removal of Detergents Most protein chemists are familiar with the classical methods for separating sample from detergent. For example, SDS can be removed by precipitation with 6 M guanidine hydrochloride (13). This should leave protein and/or peptide in the supernatant after centrifugation. However, a problem often observed with this procedure is that the compound of interest precipitates with the SDS, leaving only trace amounts of sample for analysis. Other techniques rely on the precipitation of protein. Common methods include the use of cold trichloroacetic acid (TCA) or neat acetone. If high concentrations of detergent have been used, the protein precipitation must be repeated several times for quantitative removal of the surfactant. Like the guanidine HCl method, however, the risks of losing the sample are high, especially when only picomole levels of protein are available. The presence of other solvents such as glycerol may also interfere with the procedure. In addition, these approaches usually cannot be used for the preparation of peptides. Dialysis is another classical method for removing unwanted small molecular weight components. The drawback of this technique is that large sample losses may occur, particularly if only low protein concentrations are present. In addition, this is not the method of choice for peptides or low molecular weight proteins because very low molecular weight cutoff membranes are generally not available. The detergent can also alter the molecular weight cutoff by changing the surface tension at the membrane pores. Another parameter to be considered is the micelle size of the detergent (see Table 1) which might make it impossible to separate the detergent from the sample by dialysis. Other reports have demonstrated that off-line precipitation (14-18) or solvent extraction (19) can remove SDS and other detergents. These techniques, however, are also often accompanied by a substantial loss of proteins (2, 20) due to mechanical losses or a solubility problem with the sample in aqueous media once the detergent has been removed. In summary, one can say that many of these classical methods can be used as detergent removal strategies. However, sample losses are generally high, traces of detergents may still remain, and most of these methods are not suitable for work with peptides. If only small amounts of material are available or in cases where such potential losses are of concern, the detergent is best removed via an on-line technique.

HPLC Techniques for Detergent-Containing Protein and Peptide Samples The removal of detergent from small amounts of protein and peptide samples can be a formidable problem (14-18), especially if a reversed-phase HPLC (RPC) separation is to be used for analysis or purification. When the concentration of detergent increases above a particular absolute mass on the column, peaks start to broaden (19, 20), elute in poorly defined envelopes, or elute at or near the retention time of the detergent. Although additional chromatographic steps can remove detergents, this would be time-consuming and bears the risk of sample loss. An alternative is the use of in-line pre- or trap columns. The use of hydrophobic (21-23) or ion-exchange (24-25) materials in-line with hydrophobic separating columns has been reported, but with limitations to pressure, flow rate, organic concentration or minimum volumes. New materials that overcome these limitations have been developed for in-line removal of detergents on a conventional analytical or micro LC scale. Materials are available that: 1) retain detergents but not proteins or peptides, or 2) retain peptides or proteins but not detergents. They are commercially available in cartridges and columns designed to meet the needs of microsequencing laboratories (26). They can also be packed in microcapillary columns according to published procedures (27-29). In-line detergent removal techniques can be applied to the separation and fraction collection of proteins and peptides and can be used for LC-ESI-MS applications. For example, the reduction of the amount of detergent prior to LC/ESI-MS has been achieved through the incorporation of an in-line `trap' column just before the RPC separation (30, 31).

Removal of SDS. The presence of as little as 0.005% SDS has a tremendous effect on RPC (Figure 1) (32). In response, anion-exchange materials have been used in guard columns for some years. SDS removal is accomplished through a combination of anion-exchange and hydrophilic interaction. SDS is retained on the guard column, while peptides proceed to the RPC column. Using the usual trifluoroacetic acid (TFA)-containing mobile phases, peptides are eluted with a gradient of organic solvent. When the organic level exceeds 70%, at least 95% of the SDS desorbs and is eluted to waste. The column can then be reequilibrated for the next sample. Figure 1 shows a typical application using a 20x2.1 mm SDS removal cartridge. All the detergent from a 125 µl sample containing 0.1-0.3% SDS was removed using such a cartridge column. Figure 2 illustrates that the same effect can be achieved using the same material in a self-packed microcapillary column (30). Small amounts of SDS may be retained by the RPC column, even after elution with 95% CH3CN/0.1% TFA for 60 min, which could result in a gradual shift of retention times. If this is a concern, it is advisable to use a trap method prior to RPC. This technique requires plumbing the HPLC injector in such a way that the guard column can be taken off-line during the reverse-phase separation. This will be discussed later in more detail in the section on nonionic detergent removal. The SDS removal cartridge can then be washed separately with the appropriate concentration of CH3CN. Some anion-exchange effects can be observed at low organic concentrations of the gradient. This can result in longer retention times of strongly negatively charged samples such as phosphorylated or sulfated peptides. If the pH value of the mobile phase is above 4, other peptides may interact with the anion-exchanger as well. However, as the organic solvent concentration is increased to greater than 50%, these peptides should elute in the HILIC mode (see below). Very hydrophobic peptides or proteins may also be a problem, because they may elute from the RPC system at the same time as the free SDS. In such cases, the HILIC mode, as discussed later, may again be the better technique to use.


Figure 1 Removal of SDS from a BSA digest on-line, with a 20x2.1 mm 2SDS cartridge (PolyLC, The Nest Group) upstream from a Vydac C-18 column. (Courtesy of Paul Matsudeira, Whitehead Inst., MIT)


Figure 2. Comparison of peptide separations from cytochrome C Lys-C digested (CCKCD) in the absence and presence of SDS with SDS-removal column. The column was filled with 5-µm Vydac C18 RP support and had a precolumn filled with SDS-removal resin attached to it. The upper panel shows the separation of 10 picomoles of the digest on a micro capillary column (360-µm outer, 250-µm inner diameter, 200-mm length) filled with 5-µm Vydac C18 RP support. A total of 2 µg of SDS was injected. The column was prepared as described previously (27). The flow rate was 2 µl/min. The separation of the same sample on the same column in the presence of 0.1% SDS is shown in the lower panel.


Removal of Non-Ionic Detergents. Detergents such as Triton X -100 and Tween-80 fall into the category of nonionic detergent (NID) (see also Table 1 for more examples). In the technique discussed here, the peptide or protein is retained but not the detergent. The versatility of a 10-port injector valve is used to combine a mixed-bed ion-exchange cartridge with an RPC column for separation (Figure 3). Sample is applied to the cartridge while the valve is in the inject position (the opposite of the usual arrangement). Peptides and proteins are retained by the IEX packing when loaded in 10-100 µl of 2-10% CH3CN/10 mM buffer, pH 7 (or some other pH not corresponding to pI). The neutral detergent is flushed to waste. An additional 100 µl of the solvent is used to flush residual detergent to waste. The valve is then switched in-line with the RP trap cartridge (load position) and the sample desorbed from the NID cartridge with a second buffer (e.g., 100 µl of 2-10% CH3CN/500 mM NaCl, pH 7). Proteins and peptides are now adsorbed by the RP trap cartridge while salts are flushed to waste. For on-line LC-MS, the RP trap cartridge can be washed with a third solvent (e.g., 100 µl of CH3CN/0.2% CH3COOH) to remove the non volatile salts completely. The RP cartridge is now switched in-line (inject position) with the RPC column acting in effect as the injector loop of the LC. This technique may not work for protein or peptide mixtures possessing a broad range of pI's, as it requires that they bind to a mixed-bed IEX packing. The procedure should be evaluated with a standard prior to running a real sample.

Figure 3. Schematic of arrangement of 10-port injector valve for the removal of nonionic detergents from samples on-line.



Hydrophilic Interaction Chromatography (HILIC) HILIC is another example of an on-line detergent removal technique. As with the mixed-bed NID cartridge, proteins or peptides are retained but not the detergent. It employs a decreasing (inverse) gradient of organic solvent (29, 33, 34) in which solutes elute in the order of increasing polarity. The use of volatile salts in the mobile phase suppresses ion exclusion of ionic species and insures the selectivity based on polar, not ionic differences. Unlike ion exchange, this method allows low salt or no salt elution to occur and retains longest those molecules which elute from the RPC first. With neutral peptides one may use triethylamine (TEA)/TFA and inverse organic conditions. Highly charged molecules require low amounts (e.g., 10 mM) of salt for ion suppression, and a slight salt (perchlorate, sulfate, formate, acetate) gradient (with an inverse high organic solvent gradient) to effect desorption. Figure 4 shows the separation of an electroeluted membrane protein from SDS and Coomassie blue by HILIC. If the sample contains a significant amount of salt, phase separation can occur upon contact with the mobile phase. In that case, it may be necessary to dilute the sample and to make multiple injections prior to starting a gradient. There are anecdotal data that other modes of chromatography besides HILIC can separate detergents from peptides and proteins. Cation-exchange is quite effective at retaining and resolving peptides, for example, and is commonly performed with some organic solvent in the mobile phase. When a cation-exchange HPLC column is used in place of a RPC column, peptides will be retained but (non cationic) detergents will not. If a volatile mobile phase is required, then a gradient to dilute acetic acid can be used for elution.

Figure 4. Removal of SDS, Coomassie Blue, and salts from a sample by hydrophilic interaction chromatography (52). A gradient from 70 - 0% n-propanol in 50 mM formic acid was applied to a PolyHydroxyethyl Aspartamide column (200x4.6 mm; 5 µm; 200 Å). (Courtesy of Paul Jenö, University of Basel)



Compatibility with MALDI-MS

If no in-line removal technique has been performed, the reduction of detergent concentration prior to MALDI-MS is more difficult to achieve. The use of the thin polycrystalline film sample preparation protocol of Xiang and Beavis (35) has been demonstrated to provide sufficient reduction of RTX-100 (reduced Triton X-100) signal to allow interpretation of the spectrum from an on-membrane digest (36). In the same study however, spectra from an in-gel digest sample containing Tween-20 were still dominated by the detergent, reducing the number of peptide ions assigned. Therefore, although spectra can be obtained from RTX-100 and Tween-20 detergent -containing samples by MALDI-MS, they are not ideal, as there are still significant masses which are uninterpretable.

Detergents and Gel Electrophoresis

As the last step of sample purification, laboratories often perform the separation of proteins by SDS-PAGE. This gel alone has the resolving power of separating up to 100 components. However, the combination with isoelectric focussing increases the resolution equivalent to at least 3x103 polypeptides. As we are entering the postgenome era, two dimensional electrophoresis is a technique whose time, it appears, has finally come. In order to take full advantage of these techniques, it is important to ensure that the protein sample is properly prepared prior to gel electrophoresis. Frequently asked questions are whether the presence of detergent in the sample buffer is compatible with gel electrophoresis and, if so, at what concentrations detergent can be present. In addition, sample concentrations are often low, and the investigator may have to concentrate the sample in order to obtain a detectable protein band, which can quickly lead to very high concentrations of detergent. We tested the effect of Tween-20, Triton X100, Big Chap and octyl-beta-glucopyranoside at concentrations of 0.1%, 1% and 5% on the migration of bovine serum albumin in a 10% SDS gel and did not observe any interference of these detergents with the gel electrophoresis. Following gel electrophoresis, the gel was blotted onto PVDF membrane and all protein bands were identical to the control sample which had been gel separated and blotted without detergent. However, detailed studies of the effects of different detergents on one- or two-dimensional gel electrophoresis have yet to be performed. If in doubt about the compatibility of sample buffer composition and gel electrophoresis, we suggest simply performing a test run with a variety of standard proteins reconstituted in the sample buffer used. Once it has been established that the detergent used does not interfere with the gel electrophoresis, this technique can actually be used to remove detergents altogether from the sample and should be considered for sample preparation. If low sample concentration is an issue, funnel gels (37) provide an elegant and easy way of concentrating samples, removing detergent, and performing SDS PAGE at the same time. This way, samples can be prepared for blotting and subsequent amino-terminal sequence analysis and in-gel digestion followed by peptide mapping by mass spectrometry or chromatography as described in the next paragraph.

Detergents and Their Use in Digests of Gel-Separated Proteins

Identification of components separated by electrophoresis can be achieved by either obtaining contiguous amino acid sequence (direct amino-terminal sequencing or internal sequencing) or by the more recently described methods of peptide-mass searching and peptide-sequence tagging (5, 7, 8). Many intracellular proteins are refractive to chemical amino acid sequence analysis due to a blocked amino-terminus. In order to obtain amino acid sequence information in these cases, it is necessary to cleave the protein via chemical or proteolytic means and fractionate the resulting peptides. Protocols for the digestion and improved recovery of peptide fragments from gel-separated proteins, whether from on-membrane blots or in-gel digests, have often employed detergents (2, 6, 38). The inclusion of nonionic detergents for on-membrane digestion has been shown to be crucial for the recovery of peptides in high yield and for the recovery of longer peptides for amino-terminal sequence analysis (3, 39). Fractionation of the digestion mixture is usually achieved by RP-HPLC; therefore, it is important that the detergents do not compromise the separation or interfere with the detection of the peptides. The original on-membrane digest protocol as described by Aebersold et al. (2) employed PVP-40 to block nonspecific binding sites on the membrane prior to protease digestion, a method that is compatible with MALDI-MS analysis of the eluate (4, 40). Fernandez et al. (3) demonstrated that substitution of PVP-40 with 1% RTX-100 simplified the procedure, increased the recovery of peptides, and did not interfere with the chromatography, due to the low UV absorbance RTX-100. Henzel et al. (4) described the replacement of PVP-40 with PVP-360 to overcome the same problem of interfering UV-absorbance of the detergent with the sample. In-gel digestion protocols have employed either 0.1% SDS (with trapping on a DEAE column) (20, 24) or 0.02% Tween-20 (38, 41) during both digestion and elution. Again, these protocols were designed with RPC separation of the eluates in mind, not mass spectrometric analyses, as most of the detergents used show strong interference with mass spectral analysis.

Substitution of Detergents in Mass Spectral Analyses

Three interfering effects have to be considered if detergents are used during mass spectral analysis: 1) background ions which can obscure the protein signal, 2) suppression of the sample signal, and 3) adduct formation. In addition, during electrospray ionization (ESI) mass spectrometry, a shift of the charge envelope can take place. Extensive work has been done to evaluate the compatibility of a variety of detergents on ESI mass spectrometry (42). Nonionic saccharide detergents yielded strong ESI signals without much chemical background. In contrast, SDS performed poorly. The study showed that careful selection of the detergent and the proper concentration can enhance the mass spectral analysis of proteins and peptides which are difficult to solubilize, such as membrane proteins. As expected, ionic detergents can suppress the analyte signal due to the competition between detergent and analyte ions for ESI current (42). The combination of electrospray ionization and liquid chromatography (LC-MS) does not necessarily solve the problem of detergent interference with the ionization process. Most of the neutral detergents (RTX-100, Tween-20, and NP-40) are complex mixtures of polymeric molecules that are fractionated by RP-HPLC and, coupled with efficient ionization by ESI-MS, result in many of the spectra in an LC/ESI-MS run being obscured with detergent-related ions (43).

The presence of SDS should always be avoided, or dramatically reduced, where possible. Even in on-membrane digestion protocols, the membrane is always rinsed to remove any trace of residual SDS that might subsequently be eluted with the digested peptides. The problem can be addressed by substituting the polymeric detergents with essentially monomeric detergents with one specific mass and possibly only one retention time in chromatography. Then, the known mass of the detergent can simply be ignored. Substitution of RTX-100 with octyl-ß-glucoside has been shown to be successful in on-membrane (PVDF) digestion by Pappin et al. (44) and in an extensive study by Kirchner et al. (6). Pappin et al. (44) developed a protocol solely for MALDI-MS analysis of very low quantities of gel-separated PVDF-blotted samples. The aim of the study by Kirchner et al. (6) was to determine which of sixteen nonionic detergents evaluated produced the optimum digestion and recovery of peptides and, in addition, allowed the screening of a fraction of the digest eluate by MALDI-MS prior to RP-HPLC separation. The conclusions of this study were that, when MALDI-MS was able to be incorporated into the analysis protocol, the gluco- and maltopyranoside detergents evaluated (including octyl-ß-glucoside) provided the most desirable characteristics; recovery essentially equivalent to RTX-100, strong MALDI-MS signal, and absence of detergent clusters in MALDI-MS (6, 9) (see Figure 5).

Figure 5. MALDI mass spectra of a carbonic anhydrase II (CAII)-endoproteinase LysC digest in the presence or absence of detergents using different matrix solutions. 0.4 µg of CAII was digested with LysC in Tris buffer. This was mixed with the same volume of water (Panels A and E), 1% octylglucopyranoside in 25 mM NH4HCO3/10% methanol (Panels B and F), 0.01% Tween-20 in 200 mM NH4HCO3 (Panel C and G), or 1% RTX-100 in Tris-HCl (pH 8.0) (Panels D and H). These samples (0.3 µl) were mixed with 0.3 µl [alpha]-cyano-4-hydroxycinnamic acid matrix at 33mM in 50% acetonitrile:30% methanol: 20% water (Hewlett-Packard, Palo Alto, CA)(Panels A-D), or saturated (10mg/ml) in 50% acetonitrile: 50% 0.1% TFA (panels E-H). Note, even with the 50% acetonitrile matrix there was a loss of singal in the region close to the detergent cluster (Panel H)


In an extension of Kirchner's (6) study, Gharahdaghi et al. (45) demonstrated that with the additional increase of the acetonitrile concentration to 50% in a saturated [alpha]-cyano-4-hydroxycinnamic acid matrix solution, mass analysis by MALDI-TOF of on-membrane digestion mixtures could be carried out in the presence of 1% RTX-100, 1% octylglucopyranoside and 0.2% Tween-20 if they were diluted 1:1 with an aqueous standard prior to equal parts mixing with matrix. This provides a simpler approach than the use of the thin-polycrystalline film described in the previous section. A comparison of a carbonic anhydrase II- endoproteinase LysC digest mixed with the detergents mentioned above using either commercially available matrix or the matrix solution of Gharahdaghi et al. (45) is shown in Figure 5.

It is interesting to note that the higher organic concentration of the commercial matrix did not benefit the signal obtained from either Tween-20 or RTX-100 containing solutions. The solution containing octylglucoside produced strong signals with either matrix solution. It has yet to be demonstrated whether octyl-ß-glucoside can be used with the same success with in-gel digests, but one would expect it to be similar. In-gel digestion mixtures of proteins carried out in the presence of 0.002% Tween-20 according to Hellman et al. (41) could be analyzed by LC/ESI-MS without interference of detergent clusters (11). In this work, micro spray LC/ESI-MS/MS was successfully performed at the femtomole level and provided information on up to 60% of the complete protein sequence in only one analysis (11). The strong MALDI-MS signals observed in the presence of octyl-ß-glucoside can be explained by the results of an extensive study of MALDI matrix solution conditions by Cohen and Chait (46). Their data demonstrate that octyl-ß-glucoside provides increased peptide solubility and therefore increased signal intensity for higher mass (>3000 Da) peptides and proteins in the presence of smaller peptides. Accordingly, this generates a spectrum which shows both low and high mass components (46). With regard to the analysis of octyl-ß-glucoside-containing digest mixtures by LC/ESI-MS it should be noted that characteristic contaminants of the detergent can obscure low abundance peptides (R. Aebersold, personal communication). Therefore, it would appear as though octyl-ß-glucoside is a suitable substitute for RTX-100 for on-membrane digestion protocols.

Omitting Detergents Altogether

Protocols for in-gel and on-membrane digestion have been published that do not use detergents and are specifically developed for MS compatibility. In one approach it was demonstrated that a charge-modified PVDF membrane, Immobilon-CD, allowed high recovery of peptides by either high salt or acidic organic solvents; the former compatible with LC/ESI-MS only (47), the latter with MALDI-MS (48, 49) as well as RPC separation (50). Moritz et al. (51) provided data showing an apparent recovery of 80% of the peptides from an in-gel digest of 20 pmol of phosphorylase b (compared with a solution digest) without the use of detergents. This protocol was compatible with LC/ESI-MS. The in-gel digestion protocol from Hellman et al. (41) can also be modified by omitting the addition of detergent without significant loss of peptide recovery (U. Hellman, personal communication; K. M. Swiderek, unpublished data). This could also be shown by Moritz et al. (51) using a similar in-gel digestion protocol. Instead of adding detergent, the overall yield of the proteolytic digest can be improved by reduction and alkylation of the sample prior to gel electrophoresis by standard protocols. If the protein is provided in the form of a gel band for further digestion, in-gel reduction and alkylation can still be performed, improving the yield of recovered peptides (53).


Proteins and peptides come in a wide variety of forms, and it is not feasible to analyze all of the different types of samples with one standard protocol. Although in-gel or on-membrane digestion protocols that avoid the use of detergents have been shown to be applicable to many proteins, there will be samples that require the presence of detergents for efficient recovery. Some proteins might not be affected if the detergent is removed, others may precipitate instantaneously. In some instances, the complete removal of detergent might be an impossible task since trace amounts of detergents can interact with proteins very strongly. Therefore, the history of the protein purification must be considered if the analytical techniques utilized are sensitive to the presence of detergent. All too often, the seemingly most interesting compound is finally analyzed as a detergent artifact derived from early purification steps. It is important to consider all of the protocols that are compatible with the available analytical techniques to solve a particular protein biochemical problem.


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