Nanoflow LC Research Articles

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways inChlamydomonas reinhardtii

A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.

Nanoflow liquid chromatography–tandem mass spectrometry for the characterization of intact phosphatidylcholines from soybean, bovine brain, and liver

Nanoflow liquid chromatography–electrospray ionization tandem–mass spectrometry (nanoLC–ESI–MS–MS) was applied for the characterization of intact phosphatidylcholine (PC) lipid molecules using a homemade reversed phase capillary column with a pulled tip for direct ESI at positive ion mode. Prior to the analytical column, a short capillary trapping column was utilized for on-line pre-concentration via microcross connection. Separation of intact phosphatidylcholines in the nanoflow LC column was carried out using a binary gradient elution method at 300 nL/min. The structures of the eluted PC components were determined by analysis of the typical fragment ions of PC molecules obtained from collision-induced dissociation (CID) after each precursor scan in mass spectrometry. In the current study, nanoflow LC–ESI–MS–MS analysis of PC molecules demonstrated the ability to obtain clear structural information, such as alkyl chain lengths and the degree of unsaturation with a protonated molecule ([M + H] +) and its characteristic fragment ions ([M + H RCH 2COOH] +, [M + H RCH C O] +, and [M + H-184] +). Results from the nanoflow LC–ESI–MS experiment showed the limit of detection at 3.5 fmol for the 14:0/14:0-PC standard. This technique then was applied to intact PC extracts from soybean, bovine brain, and liver without derivatization and resulted in the identification of 28, 25, and 39 phosphatidylcholines, respectively. The LC–MS–MS method has been shown to be useful for the analysis of low concentration PC molecules in biological samples.

Protein Complexes in the Archaeon Methanothermobacter thermautotrophicus Analyzed by Blue Native/SDS-PAGE and Mass Spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics: Application to the human Jurkat T-cell proteome

A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C 18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm × 200 μm I.D. for SCX, 0.7 cm × 200 μm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 μL of NH 4HCO 3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC–MS–MS run was followed. During the sample loading–SCX fractionation–desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS–MS analysis were automated. Evaluation of the technique was made with an injection of 15 μg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.

A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics.

One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.

Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins fromShewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.

Application of Micro-Electrospray Liquid Chromatography Techniques to FT-ICR MS to Enable High-Sensitivity Biological Analysis

A microbore electrospray (ESI) injection system has been adapted to our 9.4-tesla ESI FT-ICR mass spectrometer, greatly enhancing the stability and sensitivity of the system. Spray was generated from micro-ESI needles made from sharply tapered, polished fused silica capillaries of 25 to 50 μm inner diameter. Micro-ESI permits low-level sample analysis by constant infusion at sub-μL/min flow rate over a wide range of solvent conditions in both positive- and negative-ion mode. The system is flexible and allows rapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. LC/MS analyses were accomplished by replacing micro-ESI needles with capillaries packed with reverse phase retention media to permit analyte concentration and purification prior to analysis (micro-ESI/LC). A unique nano-flow LC pumping system was developed, capable of producing a true unsplit solvent gradient at flow rates below 1 μL/min. The micro-ESI/LC FT-ICR system produces mass spectra from a mixture of three neuroactive peptides at a concentration of 500 amol/μL (5 fmol each total loaded) in biological salts with baseline separation, signal-to-noise ratio of >10:1 and mass resolving power >5000. These results represent a reduction in detection limit by a factor of ∼2 × 10 6 over the best previously published LC/FT-ICR MS data.