A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope 15N. The relative abundance of the 15N- and 14N-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.     

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.     

Protein kinases in amphibian ectoderm induced for neural differentiation

Summary Ectoderm explants from early gastrula stages of Xenopus laevis were induced with a neutralizing factor. The factor was isolated from Xenopus gastrulae and partially purified by chromatography on DEAE cellulose. The ectoderm was cultured for different periods of time and then homogenized. Protein kinase activity was determined in the homogenates from induced and control explants with histone H 1 or C-terminal peptide derived from histone H 1 as substrates. The C-terminal peptide is a more specific substrate for protein kinase C, whereas histoneH 1 is a substrate for cAMP/cGMP-dependent protein kinases as well protein kinase C. With both substrates the enzyme activity increases after induction. With the C-terminal peptide as the substrate the protein kinase activity is lower, but its relative increase after induction higher. This suggests that besides cAMP/cGMP dependent protein kinases protein kinase C or related enzymes are involved in the neural induction and differentiation processes. This corresponds to previous experiments which have shown that treatment of ectoderm with phorbol myristate acetate, an activator of protein kinase C and protein kinase C related enzymes, initiates neural differentiation. Endogeneous substrates, which are more intensively phosphorylated after induction are proteins with apparent molecular weights 21 kDa and 31 kDa. Addition of protein kinase C to the induced and control homogenates abolishes the difference in the phosphorylation rate of these proteins.     

Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis

The detection and identification of protein variants and abnormally increased modified proteins are important for clinical diagnosis. We applied soft ionization mass spectrometry (MS) to analyze proteins in blood and tissues from various patients. Over the past 8 years, we diagnosed 132 cases (55 kinds) of variant proteins including hemoglobin (Hb), transthyretin (TTR), and Cu/Zn-superoxide dismutase (SOD-1), using MS as the leading technology. Of these variants, eight were new, and nine were the first cases in Japan. Some abnormal Hb cause diseases, and most of them cause erroneous levels of glycated Hb, HbA1c, i.e., a popular index of diabetes. Most of the variant TTR causes amyloidotic polyneuropathy. Variant SOD-1 causes amyotrophic lateral sclerosis. We first showed that immunoprecipitation by a specific antiserum is a reliable and simple method to prepare protein from sera and tissues for analysis by matrix-assisted laser desorption time-of-flight MS, and liquid chromatography-electrospray ionization MS (LC-ESI-MS). The use of this technology has become widespread. Using an immunoprecipitated target protein and LC-ESI-MS, we showed that the ratios of tetra-, di- and a-sialo-transferrin from two cases of congenital glycoprotein deficient syndrome were clearly distinguishable from those of control samples. We first reported a unique modified form of TTR, that is, S-sulfonated TTR, which increased markedly and specifically in three cases with molibdenum cofactor deficiency. We proposed that S-sulfonated TTR is a useful marker for screening this disease. ESI-MS was successfully used for the accurate determination of HbA1c, and we clarified the extent of discrepancies between the HbA1c value measured by conventional methods and the accurate values for samples containing various Hb variants determined by the MS method.     

A magnetic bead-based protein kinase assay with dual detection techniques

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immunochemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via noncovalent biotin–streptavidin interactions. This noncovalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immunochemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC₅₀ (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immunochemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable, and inexpensive route to the discovery of small-molecule drug leads.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Metaproteome analysis of sewage sludge from membrane bioreactors

Microbial dynamics and enzymatic activities of activated sludge processes are not completely understood yet. A better understanding about the biology is indispensable for further process optimization. Since proteins play a key role as catalysts in sludge processes, a protocol for protein extraction and analysis by 2-D PAGE was established. It is based on phenol extraction of alkaline extracts and on a subsequent precipitation with ammonium sulphate. 2-D protein patterns obtained from different sludges collected from membrane bioreactors showed--besides common spots--significant differences. Selected proteins were identified with nano-HPLC-ESI-MS/MS. All membrane biological reactor (MBR) sludge samples investigated in this study contained elastase 3A, which implies that this human serine protease is a significant constituent of municipal wastewater. Although the identification of proteins from ammonia-oxidizing bacterium Nitrosomonas europaea was expected, the detection of a protein with homology to the marine bacterium Saprospira grandis in MBR1 was surprising.     

Multivariate analysis of single quadrupole LC‐MS spectra for routine characterization and quantification of intact proteins

Modern high‐throughput proteomic platforms allow incomparable protein mixture resolution and identification. However, such sophisticated facilities are expensive and not always accessible for routine analysis of simple mixtures. In this paper, we propose a simple methodology, based on detection of intact, nondigested proteins by LC coupled to single quadrupole MS (sqLC‐MS), followed by the analysis of the resulting spectra by multivariate analysis (MA). By doing so, even large molecular weight (MW) proteins, generating complex spectra, can be characterized to a level that allows isoform discrimination, while standard algorithms, such as MS spectrum deconvolution, cannot. To demonstrate the effectiveness of the proposed approach, we have analyzed the spectra of a set of purified, intact albumins from seven different organisms (bovine, human, rabbit, rat, sheep, mouse, and pig) as a model of microheterogenous proteins, using Projection to Latent Structure Discriminant Analysis (PLS‐DA). Although these proteins are very similar (less than 1% difference in MW), sqLC‐MS/MA allowed their classification, and the identification of unknown source samples. In addition, MA allowed precise protein quantification from the same data (calibration curve R² = 0.9966). The ability to rapidly characterize and quantify proteins, together with simplicity and affordability, could make of combined sqLC‐MS/MA a routine method for the characterization of simple mixture of known proteins.     

γ-Glutamyl semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins

Reactive oxygen species are formed in the body by several natural processes and by induced oxidative stress. The reactive oxygen species may react with the various biomolecules of the body, including proteins. In order to assess the impact of oxidative damage to proteins, we have tried to identify oxidized amino acids in blood proteins which might serve as biomarkers of oxidative damage. When oxidative damage is induced into bovine serum albumin by metal-catalysed oxidation systems, the aldehyde groups formed can be derivatized by fluoresceinamine (FINH₂). Following acid hydrolysis of FINH₂-derivatized protein, two major oxidation products, γ-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS), were found and identified by HPLC and MS. Isolation and identification of oxidized amino acids from homopolymers (poly-Arg,-Pro,-Lys,-Trp or -Leu) confirmed that GGS can originate from Arg or Pro, while AAS is an oxidation product of Lys. When oxidative stress was induced in rats by treatments with t-butyl hydroperoxide or acrolein, rat plasma protein levels of GGS and AAS were found to be significantly higher compared with control rats. The AAS-content in serum albumin or in total plasma proteins collected from eight different mammalian species was found to be inversely proportional to their maximum lifespan potential. The content of AAS in plasma proteins of untreated adult rats showed a positive correlation with the age of the rat. In young rats a negative correlation with age was found for both GGS and AAS. We conclude that GGS or AAS may be useful novel biomarkers of oxidative damage to proteins in vivo.     

Proteomic analysis on the temperature‐dependent complexes in Thermoanaerobacter tengcongensis

It is generally accepted that protein complexes play an active role in avoiding the protein degradation of the thermophiles. Thermoanaerobacter tengcongensis was cultured at three different temperatures (55, 75 and 80°C) and the extracts of protein complexes were prepared. Through blue native PAGE, the changes of the relative band volumes in response to different temperatures were semi‐quantitatively compared and six temperature‐dependent bands were obtained. These bands were excised, digested with trypsin and then analyzed with MS for the identification of protein components. With the combination of the proteins identified by LC MS/MS and MALDI TOF/TOF MS, a total of 92 unique proteins were ascertained in these complexes. Besides, some protein components were examined with Western blot, which gave us insights into the survival mechanism of thermophiles. These included (i) the composition of complex at 80°C was significantly different from that at the other two temperatures; (ii) HSPs presented in all temperature‐dependent complexes; (iii) several proteins associated with the functional pathways existed in the same complexes, indicating that the complex structure provided facility for the functional efficiency.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

ICPLQuant – A software for non‐isobaric isotopic labeling proteomics

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope‐coded protein label (ICPL)‐labeled peptides on the MS level during LC‐MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time‐consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS‐identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.     

Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

γ-Glutamyl semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins

Reactive oxygen species are formed in the body by several natural processes and by induced oxidative stress. The reactive oxygen species may react with the various biomolecules of the body, including proteins. In order to assess the impact of oxidative damage to proteins, we have tried to identify oxidized amino acids in blood proteins which might serve as biomarkers of oxidative damage. When oxidative damage is induced into bovine serum albumin by metal-catalysed oxidation systems, the aldehyde groups formed can be derivatized by fluoresceinamine (FINH₂). Following acid hydrolysis of FINH₂-derivatized protein, two major oxidation products, γ-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS), were found and identified by HPLC and MS. Isolation and identification of oxidized amino acids from homopolymers (poly-Arg,-Pro,-Lys,-Trp or -Leu) confirmed that GGS can originate from Arg or Pro, while AAS is an oxidation product of Lys. When oxidative stress was induced in rats by treatments with t-butyl hydroperoxide or acrolein, rat plasma protein levels of GGS and AAS were found to be significantly higher compared with control rats. The AAS-content in serum albumin or in total plasma proteins collected from eight different mammalian species was found to be inversely proportional to their maximum lifespan potential. The content of AAS in plasma proteins of untreated adult rats showed a positive correlation with the age of the rat. In young rats a negative correlation with age was found for both GGS and AAS. We conclude that GGS or AAS may be useful novel biomarkers of oxidative damage to proteins in vivo.     

Microfluidic biochip for chemiluminescent detection of allergen-specific antibodies

Protein microarrays for allergen-specific antibodies detection were integrated in microfluidic chips, with imaging chemiluminescence as the analytical technique. This paper demonstrates the feasibility of miniaturized chemiluminescent ELISA by presenting rapid, reproducible and sensitive detection of protein antibodies using microfluidics. Three different proteins, beta-lactoglobulin, peanut lectin and human IgG were immobilized via a "macromolecules to polydimethylsiloxane elastomer (PDMS) transfer" protocol and used as capturing agent for the detection of specific antibodies. A convenient and reversible procedure was used to bond the PDMS microarray substrate to complimentary SU-8/glass microfluidic reaction chambers. The hydrodynamic behaviours of the three proteins interactions within the micro-chambers were investigated to select the most efficient flowing parameters (come to terms with the assay time and performances). The use of optimized conditions led to the concomitant detection of three specific antibodies at pM level in 300 microL and using 6 min sample incubation time. Finally, sera from allergic patients were assayed using the microfluidic device modified with apple hazelnut and pollen allergen. The results obtained compared favourably with those obtained with the classical Pharmacia CAP system.     

DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.