Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling.

Trends in increased tuberculosis infection and a fatality rate of approximately 23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

Glycogen synthesis correlates with androgen-dependent growth arrest in prostate cancer.

BACKGROUND: Androgen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied. METHODS: We investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line. RESULTS: Androgen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number. CONCLUSION: Increased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.

End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells.

Non-homologous end-joining is an important pathway for the repair of DNA double-strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser-139 in the histone variant H2AX to form gamma-H2AX. Here we report efficient in vitro end-joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end-joining is not suppressed by the PI-3 kinase inhibitor wortmannin. During the end-joining reaction H2AX is phosphorylated at Ser-139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end-joining reaction appears to be independent of H2AX phosphorylation.

Precise peptide sequencing and protein quantification in the human proteome through in vivo lysine-specific mass tagging.

Proteomics studies demand new scalable and automatable MS-based methods with higher specificity and accuracy. Here we describe an accurate and efficient method for both precise quantification and comprehensive de novo identification of peptide sequences in complex mixtures. The unique feature of this method is based on the incorporation of deuterium-labeled (heavy) lysines into proteins through in vivo cell culturing, which introduces specific mass tags at the carboxyl termini of proteolytic peptides when cleaved by certain proteases. The mass shift between the unlabeled and the deuterated lysine (lys-d4) assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides. Lys-d4 tags can also serve as internal markers in MS/MS fragment spectra when they are buried in some peptide sequences due to miscleavages. This signal specificity circumvents the mass accuracy limitations in determining particular amino acid residues for de novo sequencing. Further, this strategy of lysine-specific tagging was successfully implemented to measure the differential protein expression of human skin fibroblast cells in response to heat shock.

The dynamic alterations of H2AX complex during DNA repair detected by a proteomic approach reveal the critical roles of Ca(2+)/calmodulin in the ionizing radiation-induced cell cycle arrest.

By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca(2+)-binding proteins were found in the H2AX complex, the roles of Ca(2+) were examined. The results indicate that Ca(2+)/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

Chromatin structure and dynamics: state-of-the-art.

A meeting entitled "Chromatin Structure and Dynamics: State-of-the-Art" organized by Jordanka Zlatanova and Sanford Leuba was held at the NIH from May 8-10, 2002. It was a timely meeting and addressed our current understanding of chromatin structure, dynamics, and function.

Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures.

The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.

Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging.

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.

Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing.

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-d4) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N- and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.

A mass spectrometric "Western blot" to evaluate the correlations between histone methylation and histone acetylation.

Histone acetylation, methylation, and phosphorylation occur predominantly in the unstructured N-terminal domains or histone "tails". These modifications and others comprise a "histone code" that directly facilitates or antagonizes association of regulatory proteins with nucleosomes to mediate changes in chromatin structure and activity. Methylation of histone H3 outside of the tail region at lysine 79 has been reported for a variety of species ranging from yeast to humans and in some gene-specific cases appears to be associated with active chromatin and transcription. Whether methylation of lysine 79 is associated with other post-translational modifications of the H3 tail is unknown. Using mass spectrometric relative quantitation, a mass spectrometric "Western blot", we compare methylation at lysines 4, 9, and 79 with acetylation of human histone H3. We find that the total levels of lysine 4 and 79 methylation (combined mono-, di-, and trimethylation) in the H3 population increase with the degree of H3 tail acetylation. The total amount of lysine 4 methylation increases progressively from less than 10% in the nonacetylated H3 to greater than 90% in the penta-acetylated H3. In addition, significant levels of lysine 4 trimethylation also occur in combination with the penta-acetylated H3 species. In contrast, the level of H3 lysine 9 trimethylation is greatest for the monoacetylated species while H3 lysine 9 acetylation occurs predominantly in hyperacetylated (tetra- and penta-acetylated) H3 isoforms. Together, these results indicate that methylation of lysine 4 and 79 as well as the switch from lysine 9 methylation to acetylation are coordinated synchronously with H3 hyperacetylation as marks of active chromatin.

Amino acid residue specific stable isotope labeling for quantitative proteomics.

Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth. In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1delta cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1delta mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances.

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression.

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC(50) of 0.5 microM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor alpha in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.

Validation of DNA sequences using mass spectrometry coupled with nucleoside mass tagging.

We present a mass spectrometry (MS)-based nucleoside-specific mass-tagging method to validate genomic DNA sequences containing ambiguities not resolved by gel electrophoresis. Selected types of (13)C/(15)N-labeled dNTPs are used in PCR amplification of target regions followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis. From the mass difference between the PCR products generated with unlabeled nucleosides and products containing (13)C/(15)N-labeled nucleosides, we determined the base composition of the genomic regions of interest. Two approaches were used to verify the target regions: The first approach used nucleosides partially enriched with stable isotopes to identify a single uncalled base in a gel electrophoresis-sequenced region. The second approach used mass tags with 100% heavy nucleosides to examine a GC-rich region of a polycytidine string with an unknown number of cytidines. By use of selected (13)C/(15)N-labeled dNTPs (dCTPs) in PCR amplification of the target region in tandem with MALDI-TOF-MS, we determined precisely that this string contains 11 cytidines. Both approaches show the ability of our MS-based mass-tagging strategy to solve critical questions of sequence identities that might be essential in determining the proper reading frames of the targeted regions.

Residue-specific mass signatures for the efficient detection of protein modifications by mass spectrometry.

Currently available mass spectrometric (MS) techniques lack specificity in identifying protein modifications because molecular mass is the only parameter used to characterize these changes. Consequently, the suspected modified peptides are subjected to tandem MS/MS sequencing that may demand more time and sample. We report the use of stable isotope-enriched amino acids as residue-specific "mass signatures" for the rapid and sensitive detection of protein modifications directly from the peptide mass map (PMM) without enrichment of the modified peptides. These mass signatures are easily recognized through their characteristic spectral patterns and provide fingerprints for peptides containing the same content of specific amino acid residue(s) in a PMM. Without the need for tandem MS/MS sequencing, a peptide and its modified form(s) can readily be identified through their identical fingerprints, regardless of the nature of modifications. In this report, we demonstrate this strategy for the detection of methionine oxidation and protein phosphorylation. More interestingly, the phosphorylation of a histone protein, H2A.X, obtained from human skin fibroblast cells, was effectively identified in response to low-dose radiation. In general, this strategy of residue-specific mass tagging should be applicable to other posttranslational modifications.

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles.

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.

Large-scale quantitative proteomic study of PUMA-induced apoptosis using two-dimensional liquid chromatography-mass spectrometry coupled with amino acid-coded mass tagging.

By coupling two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) with amino acid-coded mass tagging (AACT), we have greatly increased the analytical throughput and sequence coverage of MS-based methods for proteome-wide quantitation. The dynamic range and reproducibility of this 2D-LC-AACT quantitative approach were evaluated by profiling the mixtures with different ratios of E. coli cells grown in either regular or AACT medium. A SQL-based high thoughput MASCOT data analysis tool was developed for proteomic data sorting and mining. We investigated the early stage of apoptosis by inducing the p53 upregulated modulator of apoptosis (PUMA) through the analyses of the relative ratios of the pairwise isotope signals that were originated from the control and labeled PUMA-induced cells. In 20-hour 2D-LC-MS/MS run, 480 proteins were conclusively identified, and more than half of them were quantified. A noteworthy change in the quantitative profile was that histones and a ubiquitin conjugate protein UBC9, which are involved in DNA double-strand break (DSB) repair were significantly down-regulated in the PUMA-overexpressing apoptotic cells, suggesting the detection of DSB in the apoptotic process. The quantitative profiling efficiency of this approach was compared with the gel-based quantitative analysis scheme.

Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain.

Mycobacterium tuberculosis is an infectious microorganism that causes human tuberculosis. The cell membranes of pathogens are known to be rich in possible diagnostic and therapeutic protein targets. To compliment the M. tuberculosis genome, we have profiled the membrane protein fraction of the M. tuberculosis H37Rv strain using an analytical platform that couples one-dimensional SDS gels to a microcapillary liquid chromatography-nanospray-tandem mass spectrometer. As a result, 739 proteins have been identified by two or more distinct peptide sequences and have been characterized. Interestingly, approximately 450 proteins represent novel identifications, 79 of which are membrane proteins and more than 100 of which are membrane-associated proteins. The physicochemical properties of the identified proteins were studied in detail, and then biological functions were obtained by sorting them according to Sanger Institute gene function category. Many membrane proteins were found to be involved in the cell envelope, and those proteins with energy metabolic functions were also identified in this study.

Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging.

p53-induced apoptosis plays a pivotal role in the suppression of tumorigenesis, and mutations in p53 have been found in more than 50% of human tumors. By comparing the proteome of a human colorectal cancer cell transfected with inducible p53 (DLD-1.p53) with that of the control DLD-1 cell line using amino acid-coded mass tagging (AACT)-assisted mass spectrometry, we have broadly identified proteins that are upregulated at the execution stage of the p53-mediated apoptosis. In cell culturing, the deuterium-labeled (heavy) amino acids were incorporated into the proteome of the induced DLD-1.p53 cells, whereas the DLD-1.vector cells were grown in the unlabeled medium. In high-throughput LC-ESI-MS/MS analyses, the AACT-containing peptides were paired with their unlabeled counterparts, and their relative spectral intensities, reflecting the differential protein expression, were quantified. In addition, our novel AACT-MS method utilized a number of different heavy amino acids as internal markers that significantly increased the peptide sequence coverage for both quantitation and identification purposes. As a result, we were able to identify differentially regulated protein isozymes that would be difficult to distinguish by ICAT-MS methods and to obtain a large dataset of the proteins with altered expression in the late stage of p53-induced apoptosis. The regulated proteins we identified are associated with several distinct functional categories: cell cycle arrest and p53 binding, protein chaperoning, plasma membrane dynamics, stress response, antioxidant enzymes, and anaerobic glycolysis. This result suggests that the p53-induced apoptosis involves the systematic activation of multiple pathways that are glycolysis-relevant, energy-dependent, oxidative stress-mediated, and possibly mediated through interorganelle crosstalks.

Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry.

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.