The invention provides fast and highly accurate mass spectrometer based processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

DNA diagnostics based on mass spectrometry

The mechanisms of target cell recognition and producer cell self-protection (immunity) are both important yet poorly understood issues in the biology of peptide bacteriocins. In this report, we provide genetic and biochemical evidence that lactococcin A, a permeabilizing peptide-bacteriocin from Lactococcus lactis, uses components of the mannose phosphotransferase system (man-PTS) of susceptible cells as target/receptor. We present experimental evidence that the immunity protein LciA forms a strong complex with the receptor proteins and the bacteriocin, thereby preventing cells from being killed. Importantly, the complex between LciA and the man-PTS components (IIAB, IIC, and IID) appears to involve an on-off type mechanism that allows complex formation only in the presence of bacteriocin; otherwise no complexes were observed between LciA and the receptor proteins. Deletion of the man-PTS operon combined with biochemical studies revealed that the presence of the membrane-located components IIC and IID was sufficient for sensitivity to lactococcin A as well as complex formation with LciA. The cytoplasmic component of the man-PTS, IIAB, was not required for the biological sensitivity or for complex formation. Furthermore, heterologous expression of the lactococcal man-PTS operon rendered the insensitive Lactobacillus sakei susceptible to lactococcin A. We also provide evidence that, not only lactococcin A, but other class II peptide-bacteriocins including lactococcin B and some Listeria-active pediocin-like bacteriocins also target the man-PTS components IIC and IID on susceptible cells and that their immunity proteins involve a mechanism in producer cell self-protection similar to that observed for LciA.

https://www.pnas.org/content/pnas/104/7/2384.full.pdf

Common mechanisms of target cell recognition and immunity for class II bacteriocins

Novel compositions comprised of at least one bead conjugated to a solid support and further conjugated to at least one nucleic acid and preferred methods for making the novel compositions are described. As compared to “flat” surfaces, beads linked to a solid support provide an increased surface area for immobilization of nucleic acids. Furthermore, by selecting a bead with the desired functionality, a practitioner can select a functionalization chemistry for immobilizing nucleic acids, which is different from the chemistry of the solid support.

Beads bound to a solid support and to nucleic acids

The invention relates to a microfabricated device and methods of using the device for analyzing and sorting polynucleotide molecules by size.

Method and apparatus for analysis and sorting of polynucleotides based on size

A Deubiquitinating Enzyme Encoded by HSV-1 Belongs to a Family of Cysteine Proteases that Is Conserved across the Family Herpesviridae

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

A method for the high resolution analysis of analyte ions in a time-of-flight mass spectrometer. The method consists of the generation of an intermediate time-focus plane for ions of a certain mass at a location between an ion source and an ion reflector, and then using the ion reflector to temporally focus the ions of equal mass and differing velocities which pass this plane at the same time onto a detector. For time-of-flight mass spectrometers with an ion selector, the ion selector is particularly favorable location for this intermediate plane with time focus; and with a collision cell for the collision fragmentation of the ions, the collision cell is a particularly favorable location.

Mass resolution in time-of-flight mass spectrometers with reflectors

The invention relates to the use of a time-of-flight mass spectrometer to analyze substance molecules which are ionized by laser desorption, particularly by matrix-assisted laser desorption (MALDI). In detail it relates to the process for improving mass resolution by the known method of delayed acceleration (sometimes called delayed extraction) of the ions, and devices for the performance of this method. The invention consists of using an optical device with gridless apertures for the acceleration of the ions and refocusing the ion beam divergence due to the lens effect of the apertures, by means of a lens arrangement in the drift region of the time-of-flight spectrometer. For laser light pulses, illumination, and observation, there are further lateral holes in the electrodes of the optical device.

Method of improving mass resolution in time-of-flight mass spectrometry

The invention relates to the use of a time-of-flight mass spectrometer to analyze substance molecules which are ionized by laser desorption, particularly by matrix-assisted laser desorption (MALDI). In detail it relates to the process for improving mass resolution by the known method of delayed acceleration of the ions in the space in front of the sample support plate. The invention consists of switching the potential of an intermediate electrode which is located at a short distance in front of the sample support plate, instead of switching the potential of the sample support plate itself.

Method for improved mass resolution with a TOF-LD source

The invention relates to the exact mass determination of analyte ions in time-of-flight mass spectrometers with ionization of analyte substances on sample supports by matrix-assisted laser desorption (MALDI), and with an improvement in mass resolution by delayed ion acceleration in the field between the sample support and an intermediate electrode. It particularly relates to methods for subsequent correction of the flight time values if, during the scan, an incorrect adjustment of the distance from the sample support to the nearest acceleration electrode has occurred. The invention obtains a correction parameter for the flight times of all ions in the spectrum from an internal reference substance and corrects all flight times using a quadratic correction equation, before using the calibrated mass scale for the calculation of the exact masses. The quadratic correction equation contains a control parameter which multiplicatively affects both the linear and quadratic correction term. If no better suitable reference ions are available, oligomeric ions from the matrix of the MALDI method may be used as reference ions. The method can be applied regardless of adjustment in the optimum focus range before measurement of the spectrum, if the adjustments were made in a particular manner.

Armin Holle

Papers

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

Mass resolution in time-of-flight mass spectrometers with reflectors

Method of improving mass resolution in time-of-flight mass spectrometry

Method for improved mass resolution with a TOF-LD source

Accurate mass determination with maldi time-of-flight mass spectrometers using internal reference substances