Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.     

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis

Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.     

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.     

Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate

A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.     

Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

An anomalous electrophoretic behavior of a chitinase isoform present in both grape (Vitis vinifera L.) berries and wine was observed in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. A progressive shift of the relative molecular mass M(r) of the enzyme (from approximately 30,500 up to approximately 57,700) with increasing glycol chitin concentration in the gels up to 0.1% was revealed when samples were electrophoresed under nonreducing conditions, whereas the presence of glycol chitin had no effects when samples were reduced before SDS-PAGE separation. The M(r) of other grape and wine chitinase isoforms as well as that of the chitinase from pomegranate (Punica granatum L.) fruit was unaffected by the presence of the substrate in the gel under both reducing and nonreducing conditions. Since the enzymes were inactive during the electrophoretic separation, it is likely that the retarding effect of glycol chitin observed specifically for the unreduced chitinase band from grape and wine was due to an interaction between the substrate and a chitin-binding domain different from the catalytic site, such as that typical of class I and class IV chitinases.     

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis

Substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has become a popular procedure for the separation and identification of active fractions present in enzyme mixtures due to its relative simplicity. Procedures including high-molecular-mass substrates within the gel, such as starch for identification of amylase activity, and protein substrates, including gelatin, casein, and collagen, for revealing protease activity, have been described. SDS-PAGE separation under denaturing conditions is dependent on the molecular mass of the proteins and on the effective pore size of the gels, the last factor being affected by the inclusion of high-molecular-mass substrates into the polyacrylamide matrix. In order to quantify the effect of the addition of increasing concentrations of such substrates on protein migration, starch, gelatin, and casein were included in gels in which polyacrylamide concentration was kept constant. High-molecular-mass substrates decreased migration of proteins ranging from 6.5 to 205 kDa, although the migration pattern, and thereby the accuracy of the assignation of relative molecular masses to proteins separated on those gels, was practically unaffected. The substitution of glycine, as the carrying ion, by Tricine in denaturing electrophoresis buffer systems resulted in an improvement of the migration of proteins in substrate-containing gels. Results suggested that zymograms including substrates remain a valuable procedure for the separation and the relative molecular mass assignation of active enzyme fractions.     

Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications

A general method for detecting protease activities on acrylamide or agarose gels after sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) using specific aminoacyl p-nitroanilide (NA) or peptidyl NA as substrate is described. This method is extended from the spectrophotometric assay of p-nitroaniline, which is a chromogenic product liberated by protease action on aminoacyl NA or peptidyl NA. The acrylamide gel containing protein bands was dipped directly into a solution which contained specific synthetic aminoacyl NA or peptidyl NA as a substrate or had been overlaid with an agarose gel containing the same substrate. The p-nitroaniline released on the acrylamide or agarose gel by the specific protease was diazotized with sodium nitrite and then coupled to N-(1-naphthyl)-ethylenediamine to produce distinct activity band(s). The substrates used for protease activity staining on gels were identical to those used for spectrophotometric assays. Some applications are described.     

Purification and characterization of Bacillus cereus protease suitable for detergent industry

An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.     

Electrophoretic separation of protein kinases: altered mobility with different crosslinking agents in the presence of certain detergents

Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N,N'-methylenebisacrylamide as cross-linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent-treated preparations were electrophoresed through gels which contained either 0.1% Triton X-100 or 0.1% Nonidet P-40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP-dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.     

Native horizontal ultrathin polyacrylamide gel electrophoresis of proteins under basic and acidic conditions.

The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.     

2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: application to the analysis of IgG

A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies.     

Electrophoretic characterization of heat-stable squamous cell carcinoma antigen.

The aim of this study was to investigate the heat stability of squamous cell carcinoma (SCC) antigen, a tumor-associated serine proteinase inhibitor (serpin), in tumor tissue extract by electrophoretic methods. After heat treatment at 70 degrees C for 2 h, the tumor tissue extract showed a single main protein band of 45 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which reacted with a monoclonal antibody specific for SCC antigen. The heat-stable SCC antigen was separated by two-dimensional electrophoresis (2-DE) into four spots with pI 6.4-5.9 and Mr 44500-45 000 of SCC antigen-1. Furthermore, the SCC antigen-1 still showed its inhibitory activity against a cysteine proteinase, papain, by gelatin zymography. These results suggest that heat treatment of protein sample at 70 degrees C for 2 h may be a useful method for a partial purification of SCC antigen-1 which can inhibit lysosomal cysteine proteinases such as cathepsin L, S, and K.     

Influence of Ionic Strength, pH, and SDS Concentration on Subunit Analysis of Phycoerythrins by SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often used for subunit analysis of proteins, but it is not efficient to make the α- and β-subunits of phycoerythrins separated by normal SDS-PAGE. In this research, subunit components and subunit molecular weights of four purified phycoerythrins were analyzed by SDS-PAGE. Four factors including Tris concentration, pH, ammonium persulfate (APS), and SDS concentration were studied for their effects on SDS-PAGE of phycoerythrins. It showed that these factors can influence the separation of α- and β-subunits, electrophoresis effect of γ-subunits, apparent molecular weights of subunits, and mobility of marker proteins. The α- and β-subunits separated better in the case of lower SDS concentration, lower Tris concentration, higher pH, and/or lower APS addition in separating gels. The molecular weights of α- and β-subunits increased when Tris concentration increased in a certain range. It can be concluded that factors critical to subunit analysis by SDS-PAGE are SDS concentration and ionic strength, both of which are related to critical micelle concentration of SDS and ratio of SDS monomer to micelle in SDS-PAGE system. The ratio is postulated to influence SDS-PAGE by influencing the amount of SDS bound to polypeptides and shapes of polypeptide–SDS complexes.     

聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳染色方法新探

比较了聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)银染、考染、碘化钡染色3种染色方法;提出和比较了银染-碘化钡复染和考染-碘化钡复染2种复染方法.结果表明,银染-碘化钡复染的凝胶中,未修饰蛋白条带消失,PEG修饰蛋白条带保留,游离PEG条带显色;而考染-碘化钡复染的凝胶中,未修饰蛋白、修饰蛋白和游离的PEG条带可同时显色.两种复染方法中,PEG组分的检测限均达到了0.01μg.因此,对PEG修饰蛋白体系的SDS-PAGE可先用考染或银染后再用碘化钡复染,便可在同一块凝胶上先后或同时观察到未修饰蛋白、修饰蛋白和游离PEG的情况,简化了实验操作,方便了实验结果的比较分析.     

Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification

The previously reported fluorimetric detection of sodium dodecyl sulfate (SDS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of the fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescent detection is thought to be due to the formation of a moving boundary between leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: (i) The fluorometrically detected band disappears with addition of SDS to the catholyte; (ii) band area is proportional to protein and/or SDS load; (iii) mobility of SDS-proteins differing in mass is the same at agarose concentrations up to 3%; (iv) lowering of protein mobility by increase in gel concentration and/or increase in the size of the SDS-protein leads to band disappearance. Fluorescent detection of the band is like to be nonspecific and due to the light scattering properties of a stack comprising moving boundaries of any analytes with net mobilities intermediate between SDS (or micellar SDS) and the trailing buffer constituent at their regulated very high concentrations. The steady-state stack of SDS-proteins in the size range of 14.4-45.0 kDa, and the transient stack of an SDS-protein of 66.2 kDa have lent themselves to electroelution and characterization by mass of the proteins after removal of SDS and buffer exchange using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. The possibility to form a stack of protein between leading SDS and trailing buffer anions under conditions of weak molecular sieving (open-pore gel and small-sized protein) contributes to the understanding of moving boundaries in gel electrophoresis, but in view of the narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris‐acetate polyacrylamide gels

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.     

Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.