Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells.

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 µm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Systematic evaluation of label-free and super-SILAC quantification for proteome expression analysis.

RATIONALE: Advanced implementations of mass spectrometry (MS)-based proteomics allow for comprehensive proteome expression profiling across many biological samples. The outcome of such studies critically depends on accurate and precise quantification, which has to be ensured for high-coverage proteome analysis possible on fast and sensitive mass spectrometers such as quadrupole orbitrap instruments. METHODS: We conducted ultra-high-performance liquid chromatography (UHPLC)/MS experiments on a Q Exactive to systematically compare label-free proteome quantification across six human cancer cell lines with quantification against a shared reference mix generated by stable isotope labeling with amino acids in cell culture (super-SILAC). RESULTS: Single-shot experiments identified on average about 5000 proteins in the label-free compared to about 3500 in super-SILAC experiments. Label-free quantification was slightly less precise than super-SILAC in replicate measurements, verifying previous results obtained for lower proteome coverage. Due to the higher number of quantified proteins, more significant differences were detected in label-free cell line comparisons, whereas a higher percentage of quantified proteins was identified as differentially expressed in super-SILAC experiments. Additional label-free replicate analyses effectively compensated for lower precision of quantification. Finally, peptide fractionation by high pH reversed-phase chromatography prior to LC/MS analysis further increased the robustness and precision of label-free quantification in conjunction with higher proteome coverage. CONCLUSIONS: Our results benchmark and highlight the utility of label-free proteome quantification for applications such as target and biomarker discovery on state-of-the-art UHPLC/MS workflows.

Phosphosignature predicts dasatinib response in non-small cell lung cancer.

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin ß4, a protein that mediates cell-matrix or cell-cell adhesion. The signature was validated in cross-validation and label switch experiments and in six independently profiled breast cancer cell lines. The study supports that the phosphorylation of integrin ß4, as well as eight further proteins comprising the signature, are candidate biomarkers for predicting response to dasatinib in solid tumors. Furthermore, our results show that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible and can open a new path to discovering molecular markers for response prediction.

Comparison of SILAC and mTRAQ quantification for phosphoproteomics on a quadrupole orbitrap mass spectrometer.

Advances in mass spectrometric methodology and instrumentation have promoted a continuous increase in analytical performance in the field of phosphoproteomics. Here, we employed the recently introduced quadrupole Orbitrap (Q Exactive) mass spectrometer for quantitative signaling analysis to a depth of more than 15 000 phosphorylation sites. In parallel to the commonly used SILAC approach, we evaluated the nonisobaric chemical labeling reagent mTRAQ as an alternative quantification technique. Both enabled high phosphoproteome coverage in H3122 lung cancer cells. Replicate quantifications by mTRAQ identified almost as many significant phosphorylation changes upon treatment with ALK kinase inhibitor crizotinib as found by SILAC quantification. Overall, mTRAQ was slightly less precise than SILAC as evident from a somewhat higher variance of replicate phosphosite ratios. Direct comparison of SILAC- and mTRAQ-quantified phosphosites revealed that the majority of changes were detected by either quantification techniques, but also highlighted the aspect of false negative identifications in quantitative proteomics applications. Further inspection of crizotinib-regulated phosphorylation changes unveiled interference with multiple antioncogenic mechanisms downstream of ALK fusion kinase in H3122 cells. In conclusion, our results demonstrate a strong analytical performance of the Q Exactive in global phosphoproteomics, and establish mTRAQ quantification as a useful alternative to metabolic isotope labeling.

Delayed times to tissue fixation result in unpredictable global phosphoproteome changes.

Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (<60 min) cold ischemia times did not reveal directed or predictable changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification of 791 phosphorylation sites, we found that the distribution of ratios compared to time point zero broadens with prolonged ischemia times, but these were rather undirected and diffuse changes, as we could not detect significant alterations of individual phosphosites. On the basis of our results from RPPA and LC-MS/MS analysis of rat and mouse liver tissues, we conclude that prolonged cold ischemia results in unspecific phosphoproteome changes that can be neither predicted nor assigned to individual proteins. On the other hand, we identified a number of phosphosites which were extraordinarily stable even after 360 min of cold ischemia and, therefore, may be used as general reference markers for future companion diagnostics for kinase inhibitors.

Identifying differentially regulated subnetworks from phosphoproteomic data.

BACKGROUND: Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. RESULTS: Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. CONCLUSIONS: SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data.

Life-style changes of a halophilic archaeon analyzed by quantitative proteomics.

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.

Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey.

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.

Comparative proteome analysis across non-small cell lung cancer cell lines.

Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines.

Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.

New insights into the molecular mechanisms underlying sensitivity/resistance to the atypical retinoid ST1926 in acute myeloid leukaemia cells: the role of histone H2A.Z, cAMP-dependent protein kinase A and the proteasome.

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist.

The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling. To assess the direct impact of RG7356 binding to the CD44 receptor, a global mass spectrometry-based phosphoproteomics approach was applied to freshly isolated MDA-MB-231 tumor xenografts. Results from a global phosphoproteomics screen were further corroborated by Western blot and ELISA analyses of tumor lysates from CD44-expressing tumors. Short-term treatment of tumor-bearing mice with RG7356 resulted in modifications of the MAPK pathway in the responsive model, although no effects on downstream phosphorylation were observed in a nonresponsive xenograft model. Taken together, our approach augments the value of other high throughput techniques to identify biomarkers for clinical development of targeted agents.

Disclosure of erlotinib as a multikinase inhibitor in pancreatic ductal adenocarcinoma.

A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (K(d) = 0.09-0.358 µM) than the EGFR (K(d) 0.434 µM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.

Genome-wide proteomics of Natronomonas pharaonis.

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. According to genome analysis, the theoretical proteome consists of 2843 proteins. To reach further conclusions about its cellular physiology, the cytosolic protein inventory of Nmn. pharaonis has been analyzed using MS/MS on an ESI-Q-TOF mass spectrometer coupled on-line with a nanoLC system. The efficiency of this shotgun approach is illustrated by the identification of 929 proteins of which 886 are soluble proteins representing 41% of the cytosolic proteome. Cell lysis under denaturing conditions in water with subsequent separation by SDS-PAGE prior to nanoLC-MS/MS resulted in identification of 700 proteins. The same number (but a different subset) of proteins was identified upon cell lysis under native conditions followed by size fractionation (retaining protein complexes) prior to SDS-PAGE. Additional size fractionation reduced sample complexity and increased identification reliability. The set of identified proteins covers about 60% of the cytosolic proteins involved in metabolism and genetic information processing. Many of the identified proteins illustrate the high genetic variability among the halophilic archaea.

The low molecular weight proteome of Halobacterium salinarum.

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.

Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis.

Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and that-in contrast to current knowledge-Nalpha-acetylation is common in the archaeal domain of life with 13-18% of the proteins being Nalpha-acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.

Quantitative profiling of the membrane proteome in a halophilic archaeon.

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation.

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).