Easy Import of Protein Lists and Data

One of the main objectives of the Pinpoint software is to simplify the transition from qualitative protein research and biomarker discovery to quantitative biomarker discovery and verification, and the development of verified assays. Essential to this is the transfer of panels of putative biomarkers from qualitative ion trap- or Orbitrap-based experiments to quantitative experiments on a TSQ triple quadrupole. List of proteins in FASTA format can be exported from Thermo Scientific Proteome Discoverer, SIEVE, or BioWorks software and imported directly into the Pinpoint software.

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Automated Library Searching or In Silico Digestion

Setup of quantitative experiments can be based on previously acquired data (bio-informatics based) or based on an understanding of relevant organisms, systems, and pathways of interest (hypothesis based). In either case, the first task is to determine what peptides are likely to be generated by MS/MS analysis of enzymatically digested proteins. For bioinformatics-based experiments, the Pinpoint software can be used to search previously acquired data against proprietary or commercial libraries of MS/MS spectra. The complete peptide sequence, precursor and production ion charge states, and production ion abundances obtained by library searching allow more intelligent selection of target ions for quantitation.

For hypothesis-based experiments, proteins of interest can be theoretically digested and fragmented in silico to generate the peptide sequence, charge state, and product ion abundance information that facilitates more intelligent selection of target ions.

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Automated Selection of Precursors

One of the most unique and useful capabilities of the Pinpoint software is its ability to automatically find the optimum transitions for selected reaction monitoring (SRM). It uses a proven, robust algorithm to find the most abundant precursor and product ions, which in turn provide the most reliable quantitation. The algorithm also provides highly accurate determination of optimal collision energies for the SRM transitions.

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Flexible method design to accommodate analytical requirements

The combination of a TSQ series triple quadrupole and Pinpoint software provides a multitude of options unavailable in other instrument/software combinations, options that can dramatically enhance quantitative results.

• High-resolution SRM - Most triple quadrupole mass spectrometers are limited to unit mass resolution for selection of the precursor ions. The TSQ Quantum Ultra™ and TSQ Vantage™ instruments are the only triple quadrupoles that offer higher-resolution (0.3 u FWHM) selection of precursor ions. High-resolution selected reaction monitoring (H-SRM) provides more accurate, higher-confidence quantitation. In addition, the TSQ systems can expand the isolation window to accommodate large targeted peptides dramatically increasing the detection capability and scope of targeted protein experiments. Experimental methods can also contain SRM transitions having individual resolution settings.
• Timed SRM - Timed SRM experiments (T-SRM) are another option provided by the Pinpoint software for enhancing quantitative analyses. With prior knowledge of peptide retention times, a method can be set to look for specific transitions only during the expected retention-time window. This increases the number of SRM transitions that can effectively be monitored per experiment. It also increases the dwell time (and duty cycle) for monitoring individual peptides per experiment. The result is more accurate, more sensitive quantitation. The Pinpoint software provides robust methods of building T-SRM assays using automation. Proteome Discoverer and SIEVE libraries used to build SRM assays transfers experimentally determined retention time information used to set the detection window around the targeted peptide elution profile.
• QED support - Pinpoint software also supports quantitation enhanced data-dependent (QED) scanning. QED scanning acquires periodic full-scan MS/MS spectra in addition to the SRM scans, providing confirmatory evidence that the correct peptides are being analyzed.

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Identity verification through spectral matching and probability scoring

Verification of peptide identities is essential to quantitative biomarker discovery and verification. Pinpoint software includes spectral matching and probability scoring to assure positive identification. It can use Costa Soares correlation and P-value scoring to quantitatively assess the likelihood of false positives. Strategic filtering approaches simplify data interpretation.

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Customizable reports

Experimental results are of little use if they are trapped in the data system. Pinpoint software enables the integration of discovery results with both relative and absolute quantitation studies. Results can be reported at the protein or peptide level.

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Closing the analytical loop

Discovery is frequently an iterative process. With the Pinpoint software, results can easily be exported in order to refine subsequent qualitative and quantitative experiments.

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Compatibility

Pinpoint software is compatible with BioWorks™ 3.2 software and Proteome Discoverer software, although users of BioWorks software are encouraged to consider upgrading to the power of the new Proteome Discoverer platform. Pinpoint software is also compatible with SIEVE™ software for label-free quantitative differential expression experiments.

Pinpoint software is compatible with TSQ Quantum Ultra, TSQ Quantum Access™, and TSQ Vantage triple quadrupole mass spectrometers.

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