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Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for 'bottom-up' proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows sub-second positioning of any location on a mircotiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in [micro]LC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed.

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by [alpha]-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 [micro]g of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13 500 unique analytes were detected in a single LC/MS analysis.

A totally new and independent bacterium is created with a 21 amino acid genetic code is created, which can biosynthesize an abnormal amino acid from fundamental carbon supplies and includes the amino acid in the proteins in relation with the yellowish-brown nonsense codon. Thus, the addition of any such organisms gives a chance to evaluate the result of adding new amino acids to the genetic code, and create proteins with new or increased biological tasks.

Oligomer ion compositions of

Unfolding and folding of gaseous ubiquitin ions is investigated by electron capture dissociation (ECD) mass spectrometry. ECD confirms that the 13+ ions represent separate conformers, probably with side-chain solvated alpha-helical structures.

Alternative mechanisms propose the multiplicity of routes taken by protein folding in a 'folding funnel'. This cautions against assuming that electrospray process does not alter noncovalent bonding.

For proteins of

Disulfide bonds in gaseous multiply-protonated proteins are preferably severed in the mass spectrometer by low-energy electrons, in contrast with the excitation of the ions by photons or low-energy collisions. For S-S cyclized proteins, capture of one electron can cleave both an S-S bond and a backbone bond in the same ring, or even both disulfide bonds linking two peptide chains, improving the sequence data obtainable by tandem mass spectrometry on proteins in trace quantities.

Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of [H.sup.3][PO.sup.4] and [HPO.sup.3]. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine [Beta]-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.

In previous studies, electron capture dissociation (ECD) has been successful only with ionized smaller proteins, cleaving between 33 of the 153 amino acid pairs of a 17 kDa protein. This has been increased to 99 cleavages by colliding the ions with a background gas while subjecting them to electron capture. Presumably this ion activation breaks intramolecular noncovalent bonds of the ion's secondary and tertiary structure that otherwise prevent separation of the products from the nonergodic ECD cleavage of a backbone covalent bond. In comparison to collisionally activated dissociation, this 'activated ion' (AI) ECD provides more extensive, and complementary, sequence information. AI ECD effected cleavage of 116, 60, and 47, respectively, backbone bonds in 29, 30, and 42 kDa proteins to provide extensive contiguous sequence information on both termini; AI conditions are being sought to denature the center portion of these large ions. This accurate 'sequence tag' information could potentially identify individual proteins in mixtures at far lower sample levels than methods requiring prior proteolysis.

Mass spectrometry (MS) has joined the powerful arsenal of techniques used for structural characterization of biological molecules (1-4). MS can determine the mass of the molecule and the masses of [...]

Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows subsecond positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in µLC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast eytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed. [ABSTRACT FROM AUTHOR]

Describes an automated liquid chromatography-matrix-assisted laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry platform for high-throughput proteomics. Flow chart for the protein profiling of biological samples; Automated deposition of a reversed-phase chromatography; Reaction of a lysine residue.