Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.     

Phosphoproteome profile of human liver Chang's cell based on 2-DE with fluorescence staining and MALDI-TOF/TOF-MS

Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro-Q Diamond dye, for the high-throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2-DE, then sequentially stained with Pro-Q Diamond and Coomassie Blue G-250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI-TOF/TOF-MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2-DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection.     

质谱法分析蛇毒蛋白翻译后修饰

采用SDS-PAGE分离大连黑眉蝮蛇(Gloydius Shedaoensis)蛇毒蛋白组分, Pro-Q Emerald 488糖蛋白和Pro-Q Diamond磷酸化蛋白荧光染料用于糖蛋白和磷酸化蛋白泳带染色, 采用高效液相色谱电喷雾电离串联质谱(HPLC-nESI-MS/MS)法鉴定蛋白. SDS-PAGE胶上的8条糖蛋白带被分别鉴定为L-氨基酸氧化酶、金属蛋白酶、谷氨酰环化酶、C-端缺失L-氨基酸氧化酶、纤溶酶原激活物、磷脂酶A2(PLA2)和神经生长因子; 5条磷酸化蛋白带被分别鉴定为Stejaggregin-A、PLA2、Crisp、金属蛋白酶 P-Ⅲ和Acutolysin e precursor, 与其它蛇毒来源蛋白具有一定的同源性. 为进一步验证方法的可靠性, 采用离子交换和凝胶过滤层析技术纯化得到了PLA2, Pro-Q Diamond染色结果显示PLA2被磷酸化. 研究所得结果为进一步研究蛋白质翻译后修饰对蛇毒蛋白的生物活性、结构与功能提供了依据.     

Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining

Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.     

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.     

Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye

Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.     

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.     

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.     

Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye,a fluorescent periodate Schiff-base stain

Pro-Q Emerald 488 glycoprotein stain reacts with periodic acid-oxidized carbohydrate groups, generating a bright green-fluorescent signal on glycoproteins. The stain permits detection of less than 5-18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8-16-fold more sensitive than the standard colorimetric periodic acid-Schiff base method using acidic fuchsin dye (pararosaniline). The green-fluorescent signal from Pro-Q Emerald 488 stain may optimally be visualized using charge-coupled device/xenon arc lamp-based imaging systems or 470-488 nm laser-based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro-Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer-assisted registration techniques, images may then be merged to generate differential display maps.     

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.     

N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.     

Phosphoproteome Profiling Using a Fluorescent Phosphosensor Dye in Two-Dimensional Polyacrylamide Gel Electrophoresis

We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4–5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.     

Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals

A large body of evidence has shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic inflammation. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, we and others have demonstrated that PM contains redox-active chemicals that generate inflammation through an oxidative stress mechanism. Recently, the strengths of proteomics have enabled us to demonstrate that organic DEP extracts induce a hierarchical expression pattern of oxidative stress-induced proteins in macrophages and epithelial cells. As a further extension of this work, we now employ a new phosphosensor fluorescent dye, Pro-Q Diamond, to elucidate the induction of phosphoproteins and intracellular signaling cascades that may play a role in DEP-induced inflammation. We demonstrate that DEPs induced the phosphorylation of several phosphoproteins that belong to a number of signaling pathways as well as other oxidative stress pathways. In combination with cytokine array, phosphoproteome analysis using Pro-Q Diamond allowed us to characterize the aromatic and polar chemicals of DEPs that are involved in the activation of three different mitogen-activated protein (MAP) kinase signaling pathways.     

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.     

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.     

Sensitive phosphoprotein detection in SDS‐PAGE via Anthracene Chrome Red A stain

Protein phosphorylation, one of the most important post‐translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al³⁺) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non‐phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA‐Al³⁺‐phosphoprotein complex, unlike non‐phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro‐Q Diamond stain. ACRA stain can detect 1–2 ng of α‐casein and β‐casein, 8–16 ng of ovalbumin (OVA) and κ‐casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro‐Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8–500 ng, correlation coefficient r = 0.999), α‐casein (4–500 ng, r  = 0.992), β‐casein (4–500 ng, r  = 0.996), and κ‐casein (8–500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment.     

Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis

Blue-native polyacrylamide gel electrophoresis is a powerful tool for the separation of intact membrane protein complexes mainly applied to the analysis of the enzymes of the mitochondrial oxidative phosphorylation system (OXPHOS). Combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it reveals a two-dimensional pattern showing the individual subunits of the five OXPHOS multi-enzyme complexes. This pattern is useful in the diagnostic analysis of several diseases related to disorders in the oxidative phosphorylation system. However, in order to use this method for systematic diagnostic purposes and to be able to link disease with absence or reduced expression of specific subunits, an unambiguous identification of the individual subunits is necessary. In this study, we completed this task, implementing peptide mass fingerprinting and mass spectrometric sequence analysis. In the course of these analyses, we discovered a novel variant of a cytochrome c oxidase subunit VIc.