High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

Recovery of intact proteins from silver stained gels

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.     

Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer

In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.     

Characterisation of intact recombinant human erythropoietins applied in doping by means of planar gel electrophoretic techniques and matrix-assisted laser desorption/ionisation linear time-of-flight mass spectrometry

Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-alpha, EPO-beta and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25-50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-alpha, EPO-beta and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-alpha/beta and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 +/- 0.3 kDa for EPO-alpha/beta and 36.8 +/- 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-alpha and EPO-beta, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-alpha, EPO-beta and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein.     

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS

Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.     

On-line concentration of proteins by SDS-CGE with LIF detection

We present a simple approach for on-line concentration of SDS-protein complexes by using poly(vinyl alcohol) (PVA) solution in CGE. In comparison to the coated capillary, the presence of EOF in CGE omitted the need to fill the capillaries with polymer solutions prior to the analysis. More importantly, we found that highly reproducible separation of eight proteins by 3.5% PVA was achieved between runs and without the regeneration of high bulk EOF; the RSD of migration times was less than 0.7%. To further improve the concentration sensitivity, neutral PVA was introduced into the capillary with the help of EOF to act as sieving matrix. The occurrence of stacking at the boundary between the PVA and the sample zone is mainly due to the retardation of proteins by PVA. As a result, the LODs at an S/N of 3 for SDS-protein complexes are of the order of sub-nM to several nM. For example, the LOD for BSA is 0.78 nM, which is a 91-fold sensitivity enhancement over the normal injection. In addition, our stacking method has been applied to the analyses of proteins in Escherichia coli cells. The peak for beta-galactosidase (E. coli) was observed after 0.1 microM beta-galactosidase was spiked into the E. coli samples.     

Ultrathin-layer sodium dodecyl sulfate disc electrophoresis of proteins in the range from 10 to 220 kDa in homogeneous,low-concentrated polyacrylamide gels

This study describes an ultrathin-layer sodium dodecyl sulfate (SDS) disc electrophoresis in polyacrylamide gels of a thickness of only 150 microm. By use of 2-amino-2-methyl-1,3-propanediol/glycine instead of traditional Tris/HCl buffer in the resolving phase of the gel, proteins with a wide range of molecular sizes (10 kDa to over 220 kDa) are separated in unusually low-concentrated gels (4%T, 3.3%C). 2-Amino-2-methyl-1,3-propanediol in the resolving part of the gel contributes to stabilization of the pH value at 8.8, while glycine improves destacking as well as separation of small proteins from the bulk of stacked SDS. This method combines both the advantages of conventional slab-gel electrophoresis and capillary gel electrophoresis. It is easy to apply and well suited for all further miniaturization attempts.     

Matrices for capillary gel electrophoresis--a brief overview of uncommon gels

This article gives an overview of uncommon replaceable matrices (gels) for capillary gel electrophoresis. This electrophoretic technique is useful mainly for the separation and analysis of biopolymers-nucleic acids and their fragments, and proteins/peptides. Commonly used gels are not reviewed. Those mentioned and discussed here are gels containing saccharides, newly developed acrylamide-based gels and thermoadjustable viscosity polymers, namely triblock copolymers and grafted polyacrylamide.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

Phosphoprotein electrophoresis in the presence of Fe(III) ions

Preparation of affinity polyacrylamide gels containing immobilized Fe(III) ions for the separation of proteins exhibiting metal ion binding properties is described. The presented method enables uniform distribution of immobilized metal ions in the affinity part of the polyacrylamide separating gel. Affinity gels prepared by this way are suitable to follow the effect of different concentrations of metal ions immobilized in polyacrylamide gel on a protein electrophoretic behavior. Polyacrylamide gels containing immobilized Fe(III) ions were used to study the electrophoretic behavior of two model proteins differing in their phosphate group content: chicken ovalbumin and bovine α‐casein. For the electrophoretic separation, both the native and the denaturating conditions were used.     

A new isoelectric focusing system for fast two-dimensional gel electrophoresis using a low-concentration polyacrylamide gel supported by a loose multifilament string.

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins.     

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.     

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.     

Extraction and characterization of adenovirus

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.     

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.     

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.     

Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane

The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl‐PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl‐PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono‐dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.     

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.