Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 199 records · Page 11Linked to original sources

PubMed2001

Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression.

BACKGROUND: p53 mutations occur in almost half of all soft tissue sarcomas (STS) and may contribute to multidrug resistance (MDR) in patients with STS. Doxorubicin (Dox) is one of the most active single agents in STS but is less effective in STS with p53 mutations. The effect of reintroducing wild type (wt) p53 into STS cells harboring p53 mutations on the cytotoxicity of DOX in vitro and in vivo was studied. METHODS: The following cell lines were used in this study: SKLMS-1 STS cells, which do not express wt p53; two wt p53 stable transfectant cells derived from SKLMS-1 cells; and SKLMS-1 transfectant cells from a p53 temperature-sensitive mutant that expresses wt p53 at 32 degrees C and mutant p53 at 38 degrees C. The cytotoxicity of Dox was examined by [3-(4,5-dimethylthiazzol-2-yl)-2,5-diphenltetrazolium] (MTT) and clonogenetic assay, and the effect of reintroducing wt p53 on tumor suppression by Dox was evaluated with a tumorigenicity assay. DNA fragmentation was used to detect apoptosis. MDR-1 P-glycoprotein (P-gp) expression was detected by Western blot and immunohistochemical analyses of protein levels and by Northern blot analysis of mRNA levels, respectively. The intracellular accumulation of Dox was detected by flow cytometric analysis. RESULTS: The 50% inhibitory concentration (IC(50)) of Dox for the SKLMS-1 wt p53 transfectants decreased 16-fold compared with SKLMS-1 parental cells expressing mutant p53. Colony formation of SKLMS-1 cells after Dox treatment also was inhibited by wt p53 reintroduction. The tumorigenicity of SKLMS-1 cells was inhibited by wt p53 reintroduction alone or by Dox treatment alone and was inhibited further when p53 introduction was combined with Dox treatment in severe combined immunodeficient mice. Although no difference in DNA fragmentation, Bax expression, or Bcl-2 expression was detected among wt p53 transfectants and parental SKLMS-1 cells after Dox treatment, MDR-1 P-gp expression was decreased in wt p53 transfectants compared with parental SKLMS-1 cells. Furthermore, higher intracellular accumulations of Dox were found in wt p53 transfectants than that in SKLMS-1 cells. CONCLUSIONS: Reintroduction of wt p53 into STS cells harboring p53 mutations can enhance their chemosensitivity to Dox through the inhibition of MDR-1 P-gp expression. Thus, the combination of p53 gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients with STS.

Apoptosis↗

PubMed2001

In vitro targeted killing of prostate tumor cells by a synthetic amoebapore helix 3 peptide modified with two gamma-linked glutamate residues at the COOH terminus.

Prostate-specific membrane antigen (PSMA) is a trans-membrane protein specifically expressed in LNCaP cells, malignant human prostate tissues, and the surrounding neovasculature. PSMA is a unique exopeptidase with reactivity toward poly-gamma-glutamated folates. It can sequentially remove the poly-gamma-glutamyl termini. To target prostate tumor cells, a novel procytolytic peptide was designed with a backbone consisting of an amoebapore H3 domain modified by two gamma-linked glutamate residues at the epsilon-amino group of the COOH-terminal lysine residue. The strategy behind the design of this prolytic peptide was to inactivate the lytic amoebapore H3 peptide by replacing its functionally important COOH-terminal positive charge with negatively charged groups, which in turn might be selectively removed by the PSMA exopeptidase. This peptide exhibited little cytolytic activity toward PSMA-negative cells, such as PC-3 cells. On the other hand, this peptide exhibited strong cytolytic activity toward PSMA-positive LNCaP cells in a concentration-dependent manner. The carboxypeptidase inhibitor 4,4'-phosphonicobis (butane-1,3-dicarboxylic acid) can inhibit this activity. Moreover, this peptide also exhibited cytolytic activity toward PSMA cDNA-transfected PC-3 cells.

Amino Acid Sequence↗

PubMed2001

Neonatal immunity develops in a transgenic TCR transfer model and reveals a requirement for elevated cell input to achieve organ-specific responses.

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host's spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-gamma and the splenic T cells, although unable to proliferate or produce IFN-gamma, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-gamma production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-gamma-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.

Amino Acid Sequence↗

PubMed2001

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis.

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.

Amino Acid Sequence↗

PubMed2001

Genetic mapping and DNA sequence-based analysis of deleted regions on chromosome 16 involved in progression of bladder cancer from occult preneoplastic conditions to invasive disease.

Histologic and genetic mapping with 30 hypervariable markers mapped to chromosome 16 were performed on 234 DNA samples of five cystectomy specimens from patients with invasive bladder cancer. Allelic losses of individual markers were related to microscopically identified precursor conditions in the entire bladder mucosa and invasive cancer. Their significance for the development and progression of neoplasia from in situ preneoplastic conditions to invasive disease was analysed by the nearest neighbor algorithm and binomial maximum likelihood analysis. Using this approach we identified five distinct regions of allelic losses defined by their flanking markers and predicted size as follows. p13.3(D16S418-D16S406, 1.2 cM), p13.1(D16S748-D16S287, 12.9 cM), q12 1(D16S409-D16S514, 24.0 cM), q22.1 (D16S496-D16S515, 5.4 cM), and q24 (D16S507-D16S511, 5.9 cM and D16S402-D16S413, 17.4 cM). The regions mapping to p13.1 and q24 were involved in early intraurothelial phases of bladder neoplasia such as mild to moderate dysplasia. On the other hand the deleted region mapping to p13.3 was involved in progression of severe dysplasia/carcinoma in situ to invasive bladder cancer. Testing of markers that exhibited statistically significant LOH in relation to progression of neoplasia from precursor conditions to invasive cancer on 28 tumors and voided urine samples from 25 patients with bladder cancer revealed that q12.1 showed LOH in 46.4% of tumor and 32.0% of voided urine samples. The LOH of a single marker D16S541 could be detected in approximately 28% of tumors and 20% of voided urine samples of patients with bladder cancer. These data imply that the deleted region centered around marker D16S541 spanning approximately 10 cM and flanked by D16S409 and D16S415 contains a novel putative tumor suppressor gene or genes playing an important role in the development of human bladder cancer. To facilitate more precise positional mapping and identification of pathogenetically relevant genes, we analysed of human genome contig and sequence databases spanning the deleted regions. Multiple known candidate genes and several smaller gene-rich areas mapping to the target regions of chromosome 16 were identified.

Aged↗

PubMed2001

Chelators for radioimmunotherapy: I. NMR and ab initio calculation studies on 1,4,7,10-tetra(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO4Pr) and 1,4,7-tris(carboxymethyl)-10-(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO3A1Pr).

This work describes the modification of the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (DOTA) to improve the rate of metal loading for radioimmunotherapy applications. Previous ab initio calculations predicted that the compounds 1,4,7,10-tetra(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO4Pr) and 1,4,7-tris(carboxymethyl)-10-(carboxyethyl)-1,4,7,10-tetraazacyclododecane (DO3A1Pr) have a ca. 2000-fold improvement in yttrium metal loading rates compared to those of DOTA (Jang, Y. H.; Blanco, M.; Dasgupta, S.; Keire, D. A.; Shively, J. E.; Goddard, W. A., III. J. Am. Chem. Soc. 1999, 121, 6142-6151). In this study, we report the synthesis, purification, (1)H-NMR chemical shift assignments, pK(a) values, metal loading rate measurements, and additional ab initio calculations of these two compounds. The yttrium loading rates of DO3A1Pr are approximately twice those of DOTA, at pH 4.6 and 37 degrees C. The NMR data indicates that the DO4Pr analogue forms a stable type I complex but does not form a type II complex. The new ab initio calculations performed on DO4Pr and DO3A1Pr indicate that the rate-determining step is the deprotonation of the first macrocycle amine proton, not the second proton as assumed in the previous calculations. The new calculations predict an improvement in the rate of metal loading that more closely matches the experimentally observed change in the rate.

Acetates↗

PubMed2001

Automated mass analysis of low-molecular-mass bacterial proteome by liquid chromatography-electrospray ionization mass spectrometry.

Due to the presence of a large number of proteins in cell extracts, ion chromatograms of cell extracts obtained by electrospray ionization mass spectrometry (ESI-MS) can be quite complicated. It is found that the elevated baseline in an ion chromatogram contains many protein signals. One deficiency of current commercially available LC-ESI-MS data interpretation software is found to be the lack of functional operation that allows automated mass spectral integration and interpretation over signals hidden in the baseline. This current limitation can be overcome by a technique that involves the introduction of artificial pulses to an ion chromatogram by removing the solvent mixer in the HPLC pump. These artificial pulses are treated as chromatographic peaks by the software, thereby allowing automated spectral integration over the duration of a pulse. The reliability of mass analysis from the integrated spectra is shown to be dependent on spectral interpretation parameters such as mass spectral baseline threshold. The application of this method is demonstrated for rapid detection and mass analysis of low-molecular-mass proteins from cell extracts of Escherichia coli or Bacillus globigii.

Automation↗

PubMed2001

15-deoxy-Delta 12,14-prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors.

Hepatic myofibroblasts (hMFs) play a key role in the development of liver fibrosis associated with chronic liver diseases. Apoptosis of these cells is emerging as a key process in the resolution of liver fibrosis. Here, we examined the effects of cyclopentenone prostaglandins on apoptosis of human hMFs. Cyclopentenone prostaglandins of the J series markedly reduced hMF viability, with 15-deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) being the most potent. This effect was independent of peroxisome-proliferator-activated receptors (PPARs), because PPARgamma and PPARalpha agonists did not affect hMF cell viability, and PPARgamma, the nuclear receptor for 15-d-PGJ2, was not expressed in hMFs. Moreover, 15-d-PGJ2 did not act via a cell surface G protein-coupled receptor, as shown in guanosine-5'-O-(3-thiotriphosphate) binding assays. Cell death resulted from an apoptotic process, because 15-d-PGJ2-treated hMFs exhibited condensed nuclei, fragmented DNA, and elevated caspase-3 activity. Moreover, the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethyl ketone blocked the cytotoxic effect of 15-d-PGJ2. The apoptotic effects of 15-d-PGJ2 were reproduced by H2O2 and blocked by the antioxidants N-acetylcysteine (NAC), N-(2-mercapto-propionyl)-glycine (NMPG) and pyrrolidine dithiocarbamate (PDTC). Accordingly, 15-d-PGJ2 generated rapid production of reactive oxygen species in hMFs, via a NAC/NMPG/PDTC-sensitive pathway. In conclusion, 15-d-PGJ2 induces apoptosis of human hMFs via a novel mechanism involving oxidative stress and unrelated to activation of its nuclear receptor PPARgamma. These data underline the antifibrogenic potential of 15-d-PGJ2.

Apoptosis↗

PubMed2001

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

Amino Acid Sequence↗

PubMed2001

Loss of subtelomeric sequence associated with a terminal inversion duplication of the short arm of chromosome 4.

We report on a 4(1/2)-year-old girl, who presented with multiple minor anomalies consistent with trisomy for 4p. GTG-banding identified a de novo terminal inversion duplication of distal 4p, dup(4)(p16.3p15.3). Fluorescence in situ hybridization (FISH) with a wcp4 probe confirmed the chromosome 4 origin of the additional material. FISH with a 4p subtelomere probe, D4F26, showed no signal on the dup(4) chromosome identifying a deletion of this region. Molecular analysis of 4p STS loci confirmed the subtelomeric deletion and showed loss of the paternal allele in this region. The paternal origin of the deleted region and homozygosity for one of the two paternal alleles within the region of the duplication suggests that a sister chromatid rearrangement on the paternal chromosome 4 was involved in the formation of the dup(4) chromosome. To date, the best characterized mechanisms of formation of chromosome duplications are terminal inversion duplications of 8p, which were shown to be derived from rearrangements at maternal meiosis-I. Our data show that mechanisms other than a maternal meiosis-I rearrangement can lead to the formation of terminal inversion duplications. FISH analysis with the appropriate subtelomeric probes is warranted in terminal inversion duplications to check for associated deletions.

Child, Preschool↗

PubMed2001

Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo.

BACKGROUND: The serine-threonine kinase Akt is activated by several ligand-receptor systems previously shown to be cardioprotective. Akt activation reduces cardiomyocyte apoptosis in models of transient ischemia. Its role in cardiac dysfunction or infarction, however, remains unclear. METHODS AND RESULTS: We examined the effects of a constitutively active Akt mutant (myr-Akt) in a rat model of cardiac ischemia-reperfusion injury. In vivo gene transfer of myr-Akt reduced infarct size by 64% and the number of apoptotic cells by 84% (P<0.005 for each). Ischemia-reperfusion injury decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise and fall (+dP/dt and -dP/dt). Akt activation restored regional wall thickening and +dP/dt and -dP/dt to levels seen in sham-operated rats. To better understand this benefit, we examined the effects of myr-Akt on hypoxic cardiomyocyte dysfunction in vitro. myr-Akt prevented hypoxia-induced abnormalities in cardiomyocyte calcium transients and shortening. Akt activation also enhanced sarcolemmal expression of Glut-4 in vivo and increased glucose uptake in vitro to the level seen with insulin treatment. CONCLUSIONS: Akt activation exerts a powerful cardioprotective effect after transient ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve function of surviving cardiomyocytes. Akt may represent an important nodal target for therapy in ischemic and other heart disease.

Adenoviridae↗

PubMed2001

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography.

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Amino Acid Sequence↗

PubMed2001

Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain.

The bisphosphatase activity of the hepatic bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is repressed by its kinase domain, and regulated by cAMP-dependent protein kinase (PKA)-catalysed phosphorylation. In the present study, the mechanism by which the bisphosphatase activity is repressed by the kinase domain and regulated by phosphorylation was investigated. We found that truncation of the C-terminus of the enzyme by 25, but not 20, amino acids dramatically enhanced the catalytic rate of the bisphosphatase, abrogated the inhibition by the kinase domain, and eliminated the effect of PKA-mediated phosphorylation on activity. In addition, mutation of His444-Arg-Glu-Arg to Ala-Ala-Glu-Ala had similar effects as the deletion. Moreover, the mutations also significantly affected the phosphorylation-mediated regulation of the kinase activity of the enzyme. Furthermore, the mutations altered the pH-dependence of the bisphosphatase, and the mutant bisphosphatases were more sensitive to modification by diethyl pyrocarbonate and guanidine-induced inactivation than the wild-type enzyme. Taken together, these results demonstrate that the sequence His444-Arg-Glu-Arg plays a critical role in repression of the bisphosphatase activity by both the N-terminal kinase domain and the C-terminal tail itself. These results also explain the activation of the bisphosphatase activity by PKA-catalysed phosphorylation, by suggesting that phosphorylation may relieve the inhibitory effect of the kinase domain that is mediated by the three basic residues in this sequence.

Amino Acid Sequence↗

PubMed2001

Requirement of multiple protein domains and residues for gating K(ATP) channels by intracellular pH.

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.

Adenosine Triphosphate↗

PubMed2001

Mitochondrial endonuclease G is important for apoptosis in C. elegans.

Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

Amino Acid Sequence↗

PubMed2001

From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs.

It is reasonable to propose that gene expression profiles of purified stem cells could give clues for the molecular mechanisms of stem cell behavior. We took advantage of cDNA subtraction to identify a set of genes selectively expressed in mouse adult hematopoietic stem cells (HSC) as opposed to bone marrow (BM). Analysis of HSC-enriched genes revealed several key regulatory gene candidates, including two novel seven transmembrane (7TM) receptors. Furthermore, by using cDNA microarray techniques we found a large set of HSC-enriched genes that are expressed in mouse neurospheres (a population greatly enriched for neural progenitor cells), but not present in terminally differentiated neural cells. In situ hybridization demonstrated that many of them, including one HSC-enriched 7TM receptor, were selectively expressed in the germinal zones of fetal and adult brain, the regions harboring mouse neural stem cells. We propose that at least some of the transcripts that are selectively and commonly expressed in two or more types of stem cells define a functionally conserved group of genes evolved to participate in basic stem cell functions, including stem cell self-renewal.

Animals↗

PubMed2001

Nanoliter solvent extraction combined with microspot MALDI TOF mass spectrometry for the analysis of hydrophobic biomolecules.

A nanoliter solvent extraction technique combined with microspot matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. This method involves the use of a nanoliter droplet containing organic solvents at the tip of a small capillary for extraction. The droplet is formed inside a microliter aqueous sample containing the analyte of interest. After extraction, the droplet is deposited onto a MALDI target precoated with a thin matrix layer. Since the nanoliter droplet never touches the sample container wall, any possible extraction of contaminants adsorbed on the plastic or glassware is avoided. In addition, there is no need to concentrate the organic phase after the extraction, thus avoiding any possible loss during the concentration step. The nanoliter volume can be readily deposited onto a MALDI target, producing a high analyte concentration within a microspot. Combined with microspot MALDI, this technique allows for very sensitive analysis of the extracted analyte. The performance of this technique is illustrated in several applications involving the detection of hydrophobic peptides or phospholipids. It is shown that very hydrophobic analytes can be extracted from small-volume samples containing a large amount of salts and/or more hydrophilic analytes, which tend to give dominant signals in conventional MALDI experiments. Nanoliter extraction of analyte from samples containing less than 100 nM hydrophobic analyte and over 1 microM easily ionized hydrophilic species is demonstrated. Finally, using the analysis of the ionophore valinomycin as an example, it is demonstrated that the technique is a more reliable tool for probing metal-peptide complexes than regular MALDI sample preparations.

Peptides↗

PubMed2001

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Chromatography, Liquid↗