Capillary HPLC Research Articles

Suppression of the Ligand-mediated Down-regulation of Epidermal Growth Factor Receptor by Ymer, a Novel Tyrosine-phosphorylated and Ubiquitinated Protein

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.

Monitoring of Glycoprotein Products in Cell Culture Lysates Using Lectin Affinity Chromatography and Capillary HPLC Coupled to Electrospray Linear Ion Trap-Fourier Transform Mass Spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10-1000 in conditions where the common ES Q-TOF MS detection failed.

Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector

A highly sensitive and specific method, based on capillary high performances liquid chromatography coupled with single quadrupole mass spectrometry using electrospray ionization (capillary HPLC–ESI/MS), is proposed for the identification and quantification of iridoid glycosides in the flower buds of five Lonicera species. A Zorbax SB-C18 (0.3 mm × 150 mm, 5 μm) capillary column and a gradient elution with methanol–acetonitrile–aqueous acetate acid were utilized. The most intensive electrospray ionisation signals were found in the negative ion spectra owing to CH 3COO − adducts. Eight iridoid glycosides derived from the flower buds of Lonicera species were analyzed by mass spectrometry: sweroside (IG1), 7- O-ethyl sweroside (IG2), 7-epi vogeloside (IG3), secoxyloganin (IG4), secoxyloganin 7-butyl ester (IG5), dimethyl-secologanoside (IG6), centauroside (IG7), and loganin (IG8) using combined information on retention time, the molecular ion mass and fragment ion masses. Detection limits were lower than 1.9 ng/mL in selected ion monitoring (SIM) mode and all calibration curves showed good linear regression ( r 2 > 0.9938) within test ranges. The validated method was successfully applied to analyze eight iridoid glycosides in the flower buds of five Lonicera species and provided a new basis of assessment on quality of Flos Lonicerae.

Optimization of post-column reactor radius in capillary high performance liquid chromatography: Effect of chromatographic column diameter and particle diameter

A post-column reactor consisting of a simple open tube (Capillary Taylor Reactor) affects the performance of a capillary LC in two ways: stealing pressure from the column and adding band spreading. The former is a problem for very small radius reactors, while the latter shows itself for large reactor diameters. We derived an equation that defines the observed number of theoretical plates ( N obs) taking into account the two effects stated above. Making some assumptions and asserting certain conditions led to a final equation with a limited number of variables, namely chromatographic column radius, reactor radius and chromatographic particle diameter. The assumptions and conditions are that the van Deemter equation applies, the mass transfer limitation is for intraparticle diffusion in spherical particles, the velocity is at the optimum, the analyte's retention factor, k′, is zero, the post-column reactor is only long enough to allow complete mixing of reagents and analytes and the maximum operating pressure of the pumping system is used. Optimal ranges of the reactor radius ( a r) are obtained by comparing the number of observed theoretical plates (and theoretical plates per time) with and without a reactor. Results show that the acceptable reactor radii depend on column diameter, particle diameter, and maximum available pressure. Optimal ranges of a r become narrower as column diameter increases, particle diameter decreases or the maximum pressure is decreased. When the available pressure is 4000 psi, a Capillary Taylor Reactor with 12 μm radius is suitable for all columns smaller than 150 μm (radius) packed with 2–5 μm particles. For 1 μm packing particles, only columns smaller than 42.5 μm (radius) can be used and the reactor radius needs to be 5 μm.

Quantitative Proteomics of the Archaeon Methanococcus maripaludis Validated by Microarray Analysis and Real Time PCR

For the archaeon Methanococcus maripaludis, a fully sequenced and annotated model species of hydrogenotrophic methanogen, we report validation of quantitative protein level expression ratios on a proteome-wide basis. Using an approach based on quantitative multidimensional capillary HPLC and quadrupole ion trap mass spectrometry, coverage of gene expression approached that currently achievable with transcription microarrays. Comprehensive mass spectrometry-based proteomics and spotted cDNA arrays were used to compare global protein and mRNA levels in a wild-type (S2) and mutant strain (S40) of M. maripaludis. Using linear regression with 652 expression ratios generated by both the proteomic and microarray methods, a product moment correlation coefficient of 0.24 was observed. The correlation improved to 0.61 if only genes showing significant expression changes were included. A novel two-stage method of outlier detection was used for the protein measurements when Dixon's Q-test by itself failed to give satisfactory results. The log(2) transformations of the number of peptides or isotopic peptide pairs associated with each ORF, divided by the predicted molecular weight, were found to have moderately positive correlations with two bioinformatic predictors of gene expression based on codon bias. We detected peptides derived from 939 proteins or 55% of the genome coding capacity. Of these, 60 were overexpressed, and 34 were underexpressed in the mutant. Of the 1722 ORFs encoded in the genome, 1597 or 93% were probed by cDNA arrays. Of these, 50 were more highly expressed, and 45 showed lower expression levels in the mutant relative to the wild type. 15 ORFs were shown to be overexpressed by both methods, and two ORFs were shown to be overexpressed by proteomics and underexpressed by microarray.

Simultaneous determination of biogenic monoamines in rat brain dialysates using capillary high-performance liquid chromatography with photoluminescence following electron transfer.

Simultaneous determination of biogenic monoamines such as dopamine, serotonin, and 3-methoxytyramine in brain is important in understanding neurotransmitter activity. This study presents a sensitive determination of biogenic monoamines in rat brain striatum microdialysates using capillary high-performance liquid chromatography with the photoluminescence following electron-transfer detection technique. Separation conditions were optimized by changing the concentration of an ion-interaction agent and the percentage of an organic modifier. The high concentration of ion-interaction agent enabled the amines as a class to be separated from interfering acids, but also made the separation very long. To shorten the separation time, 10% (v/v) acetonitrile was used as the organic modifier. Eight chromatographic runs during a 3-h period were analyzed in terms of retention times, peak heights, and peak widths. Chromatograms are very reproducible, with less than 1% changes in peak height over 3 h. Typical concentration detection limits at the optimum separation conditions were less than 100 pM for metabolic acids and approximately 200 pM for monoamines. The injection volume of the sample was 500 nL. Thus, the mass detection limits were less than 50 amol for metabolic acids and approximately 100 amol for monoamines. Typical separation time was less than 10 min. To validate the technique, the separation method was applied to the observation of drug-induced changes of monoamine concentrations in rat brain microdialysis samples. Local perfusion of tetrodotoxin, a sodium channel blocker, into the striatum of an anesthetized rat decreased dopamine, 3-methoxytyramine, and serotonin concentrations in dialysates. Successive monitoring of striatal dialysates at a temporal resolution of 7.7 min showed that the injection of nomifensine transiently increased dopamine and 3-methoxytyramine concentrations in rat brain dialysate.

Use of tris(2,2'-bipyridine)osmium as a photoluminescence-following electron-transfer reagent for postcolumn detection in capillary high-performance liquid chromatography.

The photoluminescence-following electron-transfer (PFET) technique, developed in our laboratory, is a sensitive chromatographic detection method for oxidizable analytes. Because the oxidations are homogeneous, the technique avoids the problem of electrode fouling. A liquid-phase oxidant reacts with the electrochemically active analytes after separation, becoming capable of photoluminescence. Laser-induced photoluminescence is measured to quantitate the analytes. Thus, the electrochemical properties of the oxidant determine the detection selectivity, and the spectroscopic properties define the sensitivity. The properties of tris(2,2'-bipyridine)osmium (1) were investigated for use as the liquid-phase oxidant in the PFET system. The redox potential of the complex is less positive than that of tris(2,2'-bipyridine)ruthenium (2); thus, on-line generation of 1(3+) by reaction with PbO2, and selective oxidation of catechols by 1(3+), was possible. The mild oxidizing power of 1(3+) led to a lower background signal (compared to 2(3+)) when mixed with acidic mobile phases. Photoluminescence from 1(2+) was much weaker than that from 2(2+); nonetheless, the system achieved subnanomolar detection limits for dopamine, 3-methoxytyramine, and serotonin. Dopamine and 3-methoxytyramine in rat brain striatal dialysates were determined before and after the injection of nomifensine. The pH of the mobile phase can govern the detection selectivity, since oxidation of most organics is accompanied by proton transfer. Reaction of 1 with catechols showed pH-dependent sensitivity resulting from pH-dependent reaction rate changes. Since the reaction rate is also temperature dependent, increased temperature at the mixer resulted in higher sensitivity. However, the noise level also increased at elevated temperature; thus, the detection limit did not improve.

Structure elucidation of deoxylutein II isomers by on-line capillary high performance liquid chromatography– 1H nuclear magnetic resonance spectroscopy

Thermal and iodine-catalyzed photochemical ( Z/ E)-isomerization of deoxylutein II [(3 R,6′ R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene, anhydrolutein I] ( 2), the dehydration product of lutein [(3 R,3′ R,6′ R)-β,ɛ-carotene-3,3′-diol] ( 4), yielded multi-component mixtures of ( Z)-isomers. By I 2-catalyzed photoisomerization, (9 Z)- 2, (9′ Z)- 2, (13 Z)- 2, (13′ Z)- 2 and (15 Z)- 2 are generated as main products. In addition, this thermodynamic-equilibrium mixture contains traces of (9 Z,9′ Z)- 2 and other (di- Z)-isomers in minor concentrations. Thirteen isomers are chromatographically separated and detected on-line by UV–vis and mass spectrometry. (all- E)-Deoxylutein II ( 2) and six of its ( Z)-configured isomers are separated by capillary HPLC (acetone-d 6/D 2O = 85:15) and detected on-line by 1H NMR spectroscopy in a microprobe. With the microprobe and the active detection volume of 1.5 μl, it is possible to perform structure elucidation with very small amounts available for various ( Z)-isomers of deoxylutein II ( 2) in the isomerization mixture.

Nitrogen-Containing Compounds in Black Pepper Oil (Piper nigrum L.)

An investigation of nitrogen-containing compounds in black pepper oil, by capillary GC, GC/MS and HPLC techniques, revealed 20 previously unreported pyrazines, pyridines and piperidines. These species were found to be present at levels ranging from 150 ppb to 30 ppm. Odor assessment revealed that 2-ethyl-3,5-dimethylpyrazine (1.5 ppm) and 3-ethyl-2,5-dimethylpyrazine (490 ppb) contributed most significantly to the overall odor.

Down-scaling narrowbore LC-ICP-MS to capillary LC-ICP-MS: a comparative study of different introduction systems

Narrowbore high performance liquid chromatography (HPLC) was scaled down stepwise to capillary HPLC employing 2.1, 1 and 0.32 mm id separation columns. Provided that each column was operated at optimum chromatographic conditions (eluent velocity, injection volume) and provided that system connection (tubing, unions) was optimal, the influence of different HPLC-ICP-MS interfaces on extra column dispersion could be investigated. Several pneumatic nebulizers i.e. PFA-ST, MCN6000, AriMist, direct injection high efficiency nebulizer (DIHEN), CEI-100 and a costume made PFA005 nebulizer were tested. It could be shown that the contribution to extra column dispersion is minimal for the DIHEN interface. As a major advantage most of the microbore and capillary hyphenations were not prone to signal suppression due to organic solvents. The 1 mm id microbore set-up employing flow rates of 60 μL min−1 offered the best compromise in terms of sensitivity, peak dispersion, resolution, peak asymmetry and compatibility with organic solvent.

Separation and quantitative analysis of coumarin compounds from Angelica dahurica ( Fisch. ex Hoffm) Benth. et Hook. f by pressurized capillary electrochromatography

A pressurized capillary electrochromatography (pCEC) method with post-column detection cell has been developed for the therapeutically important coumarins from Angelica dahurica extract. The separation of five major coumarins (xanthotoxol, osthenol, imperatorin, oxypeucedanin hydrate, byakangelicin) was optimized with respect to composition of the mobile phase, ionic strength of buffers, pH, and applied voltage. Baseline separation was achieved for the five coumarins in less than 25 min using a mobile phase of methanol–acetonitrile–phosphate buffer (pH 4.8; 15 mM) (22.5:15:62.5, v/v/v). The method showed satisfactory retention time and peak area repeatability with the first use of post-column detection cell in the pCEC instrument. Comparing to capillary high performance liquid chromatography (capillary HPLC) and conventional high performance liquid chromatography (HPLC), higher column efficiency, and shorter analysis time were achieved in pCEC. The five coumarins in the extract samples representing different stages of traditional extraction of A. dahurica were also quantitatively analyzed by pCEC. The calibration curves were linear in the range 37–129, 36–126, 12–41, 88–306, 20–69 μg/ml of the standard solutions containing the five coumarins with correlation coefficients between 0.9976 and 0.9994.

A silica-based monolithic column in capillary HPLC and CEC coupled with ESI-MS or electrospray-atmospheric-pressure laser ionization-MS

We describe the successful coupling of CEC and capillary HPLC with the recently developed atmospheric-pressure laser ionization (APLI) method. APLI is suitable for selectively and sensitively ionizing nonpolar aromatic compounds at ambient pressure for subsequent mass-selective detection. The polycyclic aromatic hydrocarbons used as analytes are first separated either by CEC on a silica-based monolithic column or by capillary HPLC. The eluent, along with a sheath flow, is volatilized by microelectrospray and then selectively ionized by excimer laser (KrF*) radiation via two-photon excitation. A QTOF-MS is used as mass-selective detector. This interface combination makes soft ionization of thermally labile nonpolar aromatic analytes possible.

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.

Tyrosine Modification Is Not Required for Myeloperoxidase-induced Loss of Apolipoprotein A-I Functional Activities

Apolipoprotein A-I (apoAI), the major protein of high density lipoprotein, plays an important role in reverse cholesterol transport via its activity as an ABCA1-dependent acceptor of cellular cholesterol. We reported recently that myeloperoxidase (MPO) modification of apoAI inhibits its ABCA1-dependent cholesterol acceptor activity (Zheng, L., Nukuna, B., Brennan, M. L., Sun, M., Goormastic, M., Settle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P. L., Ischiropoulos, H., Smith, J. D., Kinter, M., and Hazen, S. L. (2004) J. Clin. Invest. 114, 529-541). We also reported that MPO-mediated chlorination preferentially modifies two of the seven tyrosines in apoAI, and loss of parent peptides containing these residues dose-dependently correlates with loss in ABCA1-mediated cholesterol acceptor activity (Zheng, L., Settle, M., Brubaker, G., Schmitt, D., Hazen, S. L., Smith, J. D., and Kinter, M. (2005) J. Biol. Chem. 280, 38-47). To determine whether oxidative modification of apoA-I tyrosine residues was responsible for the MPO-mediated inactivation of cholesterol acceptor activity, we made recombinant apoAI with site-specific substitutions of all seven tyrosine residues to phenylalanine. ApoAI and the tyrosine-free apoAI were equally susceptible to dose-dependent MPO-mediated loss of ABCA1-dependent cholesterol acceptor activity, as well as lipid binding activity. MPO modification altered the migration of apoAI on SDS gels and decreased its alpha-helix content. MPO-induced modification also targeted apoAI tryptophan and lysine residues. Specifically, we detected apoAI tryptophan oxidation to mono- and dihydroxytryptophan and apoAI lysine modification to chlorolysine and 2-aminoadipic acid. Thus, tyrosine modification of apoAI is not required for its MPO-mediated inhibition of cholesterol acceptor activity.

Fingerprint Studies of Radix Scutellariae by Capillary Electrophoresis and High Performance Liquid Chromatography

Two methods based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) are described to establish fingerprints of Radix Scutellariae simultaneously. In order to choose an appropriate extraction method, Radix Scutellariae samples extracted by different methods were determined by HPLC. The contents of baicalin, the quality marker of Radix Scutellariae, as well as the number of peaks in the chromatograms were determined to evaluate the extraction methods. 10 batches of Radix Scutellariae collected from different regions in China were applied to establish the fingerprints. Eleven common peaks were isolated within 12 min by CE. The fingerprints obtained with HPLC consisted of 14 common peaks within 40 min. The two proposed methods demonstrated good stability and reproducibility with RSD less than 4% for relative migration time in CE and retention time in HPLC. Finally, the data from the 10 batches of Radix Scutellariae by CE and HPLC were all processed with two kinds of mathematical methods including correlation coefficient and the included angle cosine. The fingerprints of Radix Scutellariae established with CE and HPLC are suitable to identify and differentiate samples by geographical origin and can used for quality control.

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 μm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of −12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.

Evaluation of ring-opening metathesis polymerization (ROMP)-derived monolithic capillary high performance liquid chromatography columns

Novel monolithic capillary HPLC columns were prepared via ring opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 μm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens and RuCl 2(PCy 3) 2(CHPh) as initiator. Using the monolithic capillary HPLC columns, different sets of analytes (i.e. standard systems) were used for the evaluation of the monolithic columns: (i) a protein standard consisting of six proteins in the range of 5000–66 000 g/mol, (ii) an insulin–albumin standard, and (iii) a peptide standard obtained from a tryptic digest of cytochrome C. With these three different standard systems the reproducibility of synthesis in terms of separation performance proved to be 1–2% relative standard deviation in t R. Variation of polymerization parameters had a significant influence on the monolithic morphology and therefore separation efficiency and back pressure. The maximum analytical loading capacity of ROMP-derived monolithic capillary columns for albumin was found to be 30–125 ng, depending on the monomer content. Long-term stability studies showed no alteration in separation performance.

Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.