Capillary HPLC Research Articles

Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP‐70 using microprobe NMR and on‐line coupled capillary HPLC–NMR

The protein kinase ZAP-70 is involved in T-cell activation and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485-496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y-492 and Y-493 tyrosines were specifically synthesized and analyzed by (1)H/(13)C-NMR at 600 MHz using a capillary HPLC-NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 microl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 microg of the analyte were used (corresponding to ca 2 mg in classical 5-mm tubes). Capillary HPLC-NMR spectra were recorded in the stopped-flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ((1)H,(1)H-COSY-90, (1)H/(13)C-HSQC, and (1)H/(13)C-HMBC).

Detection of transferrin isoforms in human serum: comparison of UV and ICP–MS detection after CZE and HPLC separations

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L(-1) borate buffer (pH 8.4) containing 3 mmol L(-1) diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm x 50 microm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L(-1) in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP-MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP-MS instrument was used to minimize polyatomic interferences on the (56)Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02-0.04 micromol L(-1) Tf for HPLC-ICP (ORS)-MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S(2), S(3), S(4), S(5), and S(6)) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.

Alternative high-performance liquid chromatographic peptide separation and purification concept using a new mixed-mode reversed-phase/weak anion-exchange type stationary phase

This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversed-phase/weak anion-exchange (RP/WAX) type stationary phase. The RP/WAX is based on N-(10-undecenoyl)-3-aminoquinuclidine selector, which is covalently immobilized on thiol-modified silica particles (5 μm, 100 Å pore diameter) by radical addition reaction. Remaining thiol groups are capped by radical addition with 1-hexene. This newly developed separation material contains two distinct binding domains in a single chromatographic interactive ligand: a lipophilic alkyl chain for hydrophobic interactions with lipophilic moieties of the solute, such as in the reversed-phase chromatography, and a cationic site for anion-exchange chromatography with oppositely charged solutes, which also enables repulsive ionic interactions with positively charged functional groups, leading to ion-exclusion phenomena. The beneficial effect that may result from the combination of the two chromatographic modes is exemplified by the application of this new separation material for the chromatographic separation of the N- and C-terminally protected tetrapeptide N-acetyl-Ile-Glu-Gly-Arg- p-nitroanilide from its side products. Mobile phase variables have been thoroughly investigated to optimize the separation and to get a deeper insight into the retention and separation mechanism, which turned out to be more complex than any of the individual chromatography modes alone. A significant anion-exchange retention contribution at optimal pH of 4.5 was found only for acetate but not for formate as counter-ion. In loadability studies using acetate, peptide masses up to 200 mg could be injected onto an analytical 250 mm × 4 mm i.d. RP/WAX column (5 μm) still without touching bands of major impurity and target peptide peaks. The corresponding loadability tests with formate allowed the injection of only 25% of this amount. The analysis of the purified peptide by capillary high-performance liquid chromatography (HPLC)-UV and HPLC–ESI-MS employing RP-18 columns revealed that the known major impurities have all been removed by a single chromatographic step employing the RP/WAX stationary phase. The better selectivity and enhanced sample loading capacity in comparison to RP-HPLC resulted in an improved productivity of the new purification protocol. For example, the yield of pure peptide per chromatographic run on RP/WAX phase was by a factor of about 15 higher compared to the standard gradient elution RP-purification protocol.

Pseudouridine detection improvement by derivatization with methyl vinyl sulfone and capillary HPLC–mass spectrometry

A method is presented for improved detection of pseudouridine in nucleoside mixtures based on the specific derivatization with methyl vinyl sulfone followed by analysis by capillary HPLC-mass spectrometry. Reaction conditions were optimized in order to obtain the best yield and specificity. The method was successfully applied to different nucleoside mixtures.

Cholesterol-lowering effects of dietary blue lupin (Lupinus angustifolius L.) in intact and ileorectal anastomosed pigs

The present study was undertaken to investigate the effect of cholesterol-enriched casein (CAS) and blue lupin seed (BL) diets on the cholesterol metabolism of intact (INT) and ileorectal anastomosed (IRA) pigs. For 3 weeks, four groups of six pigs were allocated to the treatments (CAS-INT, CAS-IRA, BL-INT, and BL-IRA). Diet-induced hypercholesterolemia was inhibited by the BL through a substantial decrease in plasma LDL-cholesterol. The BL also reduced liver esterified and total cholesterol, increased hepatic LDL receptor synthesis and HMG-CoA reductase activity, and stimulated intestinal bile acid reabsorption. The neutral sterol output was higher in BL- than in CAS-fed pigs. The bile acid output was lower in IRA than in INT pigs. Surgery also prevented steroid microbial transformation, but it did not influence plasma cholesterol levels. These results suggest that the hypocholesterolemic effect of the BL, compared with the CAS, is attributable to impaired intestinal cholesterol absorption, probably involving increased bile acid reabsorption and higher contents of dietary phytosterols, both factors that reduce the micellar solubilization of cholesterol. Furthermore, according to our data, the contribution of the large intestine to cholesterol metabolism is very weak.

Feasibility of assessment of regulatory lipids in breath condensate as potential presymptomatic harbingers of pulmonary pathobiology

Regulatory lipids from the airway surface readily form aerosols that can be recovered non-invasively by cooling expired breath to form breath condensate (BC). Regulatory lipids have been detected previously utilizing enzyme-linked-immunosorbent serologic assay (ELISA). Here we test the feasibility of assessment of regulatory lipids in BC by mass spectrometry so presently unknown lipid regulatory components can be detected without addition of specific antibodies as in the ELISA procedure. Baseline regulatory lipids were detected in > pg/mL BC in control animals or human lung tissue culture cells. In nearly every case animals exposed to toxins or infectious bacteria showed increases in the BC regulatory components. Lipids were recovered from BC by solid phase extraction. Phosphatidylcholine (PC) based lipids were detected as the progenitor (parent) ions of isomers that fragmented in producing product positive ions at m/ z 184 (of phosphocholine) in tandem MS using capillary HPLC and electrospray ionization. BC eicosanoids such as prostaglandins, thromboxane, and isoprostanes require capillary gas chromatography for separation and detection that necessitates methoximation, pentafluorobenzyl (PFB) ester formation, and trimethyl silylation of hydroxyls prior to gas chromatography/ion trap tandem mass spectrometry of negative ions after chemical ionization (NICI). Tetradeuterated internal standards were utilized for quantitation with the GC/NICI/MS. Changes in concentrations of lipids and eicosanoids were observed in piglets, and rats exposed to aerosolized 100 μg/kg lipopolysaccharide (LPS), or 50 μg/kg and 150 μg/kg aerosolized Staphylococcal enterotoxin B (SEB) in BC as well as in human THP-1 cell culture cell supernatants and bronchoalveolar lavage (BAL) samples in rats. Responses of the molecular species of phosphatidylcholines (PCs), platelet activating factors (PAFs) and specific eicosanoids correlated to the toxin and bacterial infections suggesting that patterns of differential responses could be detected with further experimentation. Initial targets included prostaglandins (PGE 2, PGF 2α), thromboxane (TXB 2), and prostacyclin (as 6-Keto PGF 1α) that show differential responses to inflammation, the leukotriene (LTB 4) and PGD 2 for allergic responses, isoprostanes (8- iso-PGF 2α) for free radical oxidative stress responses, and HETEs for differential lipoxygenase activities. PAFs and lysoPAFs have been shown to increase with inflammation and in the feasibility experiments reported here. Preliminary studies show pulmonary responses of piglets to intrathecal exposure of toxicants (LPS and SEB) or infections with Actinobacillus pleuropneumoniae induce increased levels of lipids and two eicosanoids with the suggestion that differential patterns might be detected with expanded testing. Preliminary experience indicates numerous other eicosanoids were available for assay in BC. This suggests an important potential application of BC to observe a wide array of factors to establish comprehensive profiles for physiological and pathophysiological states. Ultimately this technique could be used as a non-invasive possibly presymptomatic assessment of pulmonary pathobiology.

Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry

Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Liquid chromatography‐electron ionization mass spectrometry: Fields of application and evaluation of the performance of a Direct‐EI interface

A comprehensive evaluation and a thorough discussion of the fields of possible applications of the Direct-EI interface are described in this review. Direct-EI allows the direct introduction of the effluent from a capillary HPLC column into the electron ionization (EI) ion source of a mass spectrometer. Thanks to the reduced liquid intake and an in-source nebulizer, the interfacing process occurs smoothly and entirely into the ion source. No intermediate interfacing mechanisms of any sort are interposed between the column and the mass spectrometer, thus circumventing any undesired sample loss and minimizing the effort for instrument modification. Theoretically, any GC-MS system can be converted into an LC-MS for EI amenable compounds. Several parameters, crucial for a successful integration of liquid chromatography and mass spectrometry, have been considered in the evaluation of the functioning of such an interface: limit of detection, linearity of response, reproducibility, and chromatographic compatibility. Different mobile phases, also containing non-volatile buffers, were taken into account, demonstrating an outstanding separation flexibility. The entire set of experiments was carried out at different flow rates and temperatures of the ion source. The interface behavior was also tested in real world applications, with mixtures of pesticides, hormones, nitro-PAH, and endocrine-disrupting compounds, allowing picogram level detection and the possibility to record library-matchable, readily interpretable electron ionization mass spectra, for prompt compound characterization and confirmation.

A Method for Quantitating the Intracellular Metabolism of AZT Amino Acid Phosphoramidate Pronucleotides by Capillary High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry

A methodology has been developed for the analysis of the intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT) amino acid phosphoramidates utilizing reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC/ESI(-) -MS). The presented work demonstrates the potential of capillary LC/MS and LC/MS/MS to identify and quantitate the cellular uptake and metabolism of nucleoside phosphoramidate. Significant intracellular amounts of D- and L-phenylalanine methyl ester or D- and L-tryptophan methyl ester AZT phosphoramidates were observed for human T-lymphoblastoid leukemia (CEM) cells incubated for 2 and 4 h with the prodrugs. AZT-MP was the primary metabolite observed for human T-lymphoblastoid leukemia (CEM) cells. In this paper, the details of using LC/MS to analyze AZT amino acid phosphoramidates in biological samples are discussed. LC/MS is an efficient method for analyzing multiple samples containing several analytes in a short period of time. The method also provides high selectivity and sensitivity, and requires minimal sample preparation. This approach should be broadly applicable for the analysis of the intracellular metabolism of nucleoside prodrugs and pronucleotides.

Intact-protein-based High-resolution Three-dimensional Quantitative Analysis System for Proteome Profiling of Biological Fluids

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

Hyphenation of capillary high-performance liquid chromatography to microcoil magnetic resonance spectroscopy—determination of various carotenoids in a small-sized spinach sample

The development of miniaturized hyphenated systems such as capillary high-performance liquid chromatography–and nuclear magnetic resonance spectroscopy (HPLC–NMR) remains challenging in the field of structure elucidation. In combination with a highly specific sample preparation technique, matrix solid-phase dispersion (MSPD), and a highly selective C 30 reverse phase HPLC–NMR enables the identification of small amounts of natural compounds. Here, the investigation of five carotenoids in a standard solution and two carotenoids from a spinach sample demonstrate the potential of this new development. The separation of the carotenoids is performed with self-packed fused-silica capillaries with a binary solvent gradient consisting of acetone and water. The miniaturized system allows the use of fully deuterated solvents for on-line HPLC–NMR coupling. The 1H NMR spectra of the various carotenoids obtained in stopped-flow mode gave a high signal-to-noise ratio with a sample amount in the low nanogram range. All necessary parameters for structure elucidation such as multiplet structure, coupling constants and integration values can be detected unambiguously.

Metabonomic Studies Comparing Capillary and Conventional HPLC-oa-TOF MS for the Analysis of Urine from Zucker Obese Rats

The application of capillary HPLC and orthogonal acceleration (oa) TOF-MS to a metabonomic investigation of the urinary metabolic profiles of samples obtained from male and female Zucker rats is described. The results obtained using capillary HPLC for performing the chromatographic separation are also compared with the analysis of the same samples by conventional HPLC. Capillary HPLC-MS gave increased sensitivity (ca. 100 fold) despite using a fraction of the sample volume consumed by HPLC (0.5 μL vs 10 μL). Capillary HPLC-MS also provided a greater peak count (ca. 3000 ions compared to 1500 for HPLC) for the same analysis time compared to conventional HPLC-MS. These data were processed using partial least squares discriminant analysis (PLS-DA)) enabling markers of diurnal variation to be detected for urine samples from both male and female rats.

Analysis of Supercritical Thermohydrolysis of Aniline‐Water Solution using Capillary Electrophoresis and HPLC: a Comparison

In this paper, an application of capillary electrophoresis and high‐performance liquid chromatography for aniline thermohydrolysis in an environment of supercritical water is presented.

Determination of Total Phenols in Environmental Waters by Capillary-HPLC with U.S.E.P.A. Classified Eleven Priority Pollutant Phenols after Nitrosation and Their Visible Spectrophotometric Detection

The determination of total phenols was accomplished by capillary-high performance liquid chromatography (capillary-HPLC) after nitrosation of the U.S.E.P.A. classified 11 priority pollutant phenols, using the nitrosated parent phenol (POHNO) as a reference for calibration. The optimum mobile phase composition for this analysis was found by examining the effect of changing the percentage of acetonitrile (MeCN) in the mobile phase on retention factors (k values) and peak intensities. As MeCN percentage was increased, k values were reduced and peak intensities were generally increased. From the results obtained, it was found that the optimum mobile phase was 90%(v/v) MeCN solution at pH 8.0, the detection wavelength of 400 nm, and a detection limit (D.L., concentration at signal to noise ratio (S/N) of 3.0) of 4.5 <TEX>${\times}$</TEX> <TEX>$10^{-7}$</TEX> M. In addition, 10 of the 11 phenols present in mineral or waste water were separated after the nitrosation by capillary-HPLC. The optimum mobile phase for separation was a 40%(v/v) MeCN solution at pH 5.0.

Effect of temperature on the separation of chlorophenoxy acids and carbamates by capillary high-performance liquid chromatography and UV (or diode array) detection

In this work, the effect of temperature in isothermal and programmed modes on several chromatographic parameters such as retention factor, selectivity, resolution and plate number has been discussed. A critical comparison of isocratic/isothermal, gradient/isothermal and isocratic/program temperature modes has been made. Two representative families of pesticides have been selected for this study. One includes ionisable chlorophenoxy acids and two of their esters, some of which show similar polarities. The other one contains several weakly polar carbamates. Analysis was carried out using a reversed-phase capillary high-performance liquid chromatography (HPLC) system and focusing technique with UV or diode array detection (DAD).

Online preconcentration of thyrotropin-releasing hormone (TRH) by SDS-modified reversed phase column for microbore and capillary high-performance liquid chromatography (HPLC)

Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-amide) is an important tripeptide existing in biological systems at low concentrations. It is a fairly hydrophilic peptide, cationic in acidic solutions. Preconcentration online before reversed phase chromatography separation can enhance concentration detection limits of hydrophobic, but not hydrophilic species. The hydrophilic TRH can be preconcentrated using a reversed phase precolumn charged with sodium dodecyl sulfate (SDS). The separation also uses SDS. The preconcentration is effective for a microbore system, achieving detection limit of 250 pM for a sample size of 500 μl with electrochemical detection of the biuret complex formed post column. Preconcentration using an online precolumn is also effective in packed capillary high-performance liquid chromatography (HPLC) with a detection limit of 3 nM in 24 μl.

Advances in analytical methodology for bioinorganic speciation analysis: metallomics, metalloproteomics and heteroatom-tagged proteomics and metabolomics

The recent developments in analytical techniques capable of providing information on the identity and quantity of heteroatom-containing biomolecules are critically discussed. Particular attention is paid to the emerging areas of bioinorganic analysis including: (i) a comprehensive analysis of the entirety of metal and metalloid species within a cell or tissue type (metallomics), (ii) the study of the part of the metallome involving the protein ligands (metalloproteomics), and (iii) the use of a heteroelement, naturally present in a protein or introduced in a tag added by means of derivatisation, for the spotting and quantification of proteins (heteroatom-tagged proteomics). Inductively coupled plasma mass spectrometry (ICP MS), used as detector in chromatography and electrophoresis, and supported by electrospray and MALDI MS, appears as the linchpin analytical technique for these emerging areas. This review focuses on the recent advances in ICP MS in biological speciation analysis including sensitive detection of non-metals, especially of sulfur and phosphorus, couplings to capillary and nanoflow HPLC and capillary electrophoresis, laser ablation ICP MS detection of proteins in gel electrophoresis, and isotope dilution quantification of biomolecules. The paper can be considered as a followup of a previous review by the author on a similar topic (J. Szpunar, Analyst, 2000, 125, 963).

Protein profile of silkworm midgut of fifth-instar day-3 larvae

Proteomic analysis was performed on the midgut of fifth-instar day-3 female silkworm (Bombyx mori, strain p50) larvae. Though silkworm genome analysis has not yet been completed, the Drosophila genome and silkworm expression sequence tag (EST) data were applied to the analysis of the midgut proteins in the database (DB) for identification. The spots, which were excised manually from two-dimensional electrophoresis (2-DE) gels, were treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry (MS) after proteolysis using a trypsin. Nearly 60% of the proteins analyzed by MS were identified. These included many kinds of cytoskeleton proteins, ATPase, and chaperonins.

Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.