ProtalizerTM DDA software platform
Accurate Answers in Proteome-Wide Research Studies
Discover the underlying proteins and PTMs differentially regulated in your samples today with the turnkey solution Protalizer DDA. This tool is a perfect solution for any label-free DDA application ranging from therapeutic antibody characterization, protein-protein interaction (PPI) studies, and large-scale total lysate quantitative proteomics.
Discover the underlying proteins and PTMs differentially regulated in your samples today with the turnkey solution Protalizer DDA. This tool is a perfect solution for any label-free DDA application ranging from therapeutic antibody characterization, protein-protein interaction (PPI) studies, and large-scale total lysate quantitative proteomics.
Calibration of Parent and Fragment Ion m/z
Obtaining correct identification and quantification results requires correcting observed m/z values to the most accurate values possible. Protalizer DDA does this by applying an m/z dependent calibration on all precursor and fragment ion m/z values in each file based on observed errors in each 25 m/z range. Calibrated peak data is then researched at a fixed FDR rate based on a target-decoy search.
Obtaining correct identification and quantification results requires correcting observed m/z values to the most accurate values possible. Protalizer DDA does this by applying an m/z dependent calibration on all precursor and fragment ion m/z values in each file based on observed errors in each 25 m/z range. Calibrated peak data is then researched at a fixed FDR rate based on a target-decoy search.
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ProtalizerTM DDA reduces m/z error by 4-fold.
Data on the right shows HeLa tryptic digests analyzed on a Bruker Impact II QTOF with a 90 minute reversed-phase gradient searched against the human Swiss-Prot database with and without applying m/z calibration. The raw data were downloaded from ProteomeXchange accession number PXD001592. 
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Error Bars = Standard Deviation
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Sensitive Detection of PTMs
Accurately calibrated spectra are searched by the X! Tandem pile driver search engine – one of the most widely vetted tools in proteomic research. Unique features included in the Protalizer DDA software suite include:
Accurately calibrated spectra are searched by the X! Tandem pile driver search engine – one of the most widely vetted tools in proteomic research. Unique features included in the Protalizer DDA software suite include:
- Real-time monitoring of 12 Quality metrics
- FDR filtering to target rate based on a target-decoy reversed database search
- Submission of samples in batch format
- Support for combining identifications from multiple sample fractions (e.g. SDS-PAGE, strong-cation-exchange, etc.)
- Point-and-click support for all the modifications listed below and ability to specify other modifications not listed
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MS1 Chromatogram Relative Quantification Across Biological Conditions Compared
Peptides identified in each sample are matched to MS1 chromatograms. Then to avoid missing quantitative data, peptides not identified in each sample are searched for with normalized retention times based on the retention time drift of MS1 chromatograms of common peptides detected in multiple samples. Due to the variation of DDA methods in different laboratories, the software allows users to adjust various settings in the MS1 chromatogram extraction algorithm to obtain the best results with any dataset (e.g., the minimum number of consecutive MS1 scans a peptide precursor must be detected, etc.). Monitoring the efficacy of these changes is facilitated by Quality metric #7 reported by the program that shows the percentage of identified peptides that were able to be matched to chromatograms. Rapid Point-and-Click Review of MS1 Quantification Results
A major problem verifying differences by visual inspection in large-scale studies are that thousands of proteins are often quantified resulting in large numbers of differentially regulated proteins across the sample conditions compared. To solve this issue, ProtalizerTM DDA generates chromatogram archives of every peptide compared across samples. These digital chromatograms reduce the time needed to verify findings compared to other visualization tools, are created in HTML to promote use by non-proteomic specialists, and allow querying all the peptides for a specific protein of interest by protein name rather than through numerous mass-to-charge ratio inquiries. Furthermore, to address technical artifacts caused by ion suppression and instrument performance variability, the chromatograms include a co-eluting (or closely-eluting) ‘endogenous reference peptide’ as a quantification quality control for each peptide quantified. These references are selected based on having the most consistent concentration across the samples analyzed, and provide retention time-specific intensity normalization that reduces the peptide coefficient of variation. Protalizer DDA rapid MS1 XIC quantification view. Top panel shows a peptide quantified from 10 kDa heat shock protein, FLPLFDR with the monoisotopic, 1C13, and 2C13 traces and the bottom panel shows the monoisotopic traces only for five co-eluting endogenous reference peptides.
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Statistical Analysis
Protein relative abundance across different biological conditions are preferentially quantified by peptides specific to the assigned protein that were detected in each sample compared. Statistical significance of protein differences can be determined with the software for comparisons with up to 10 different biological conditions.
Protein relative abundance across different biological conditions are preferentially quantified by peptides specific to the assigned protein that were detected in each sample compared. Statistical significance of protein differences can be determined with the software for comparisons with up to 10 different biological conditions.
Reporting/readouts
Additional files generated from each analysis:
Additional files generated from each analysis:
- Rawseq files for identifications made in each sample
- Quality report that allows objectively determining dataset reliability with 12 evaluated metrics
- Config report that shows the user-selected and default parameters used to analyze data
- Peptide quant summary shows the intensity, retention time, and normalized abundance for every peptide
- Peptide-ratios-per-reference contains the normalized abundance for each peptide using each endogenous reference
- Protein quant summary shows the relative abundance of proteins across biological groups compared, P-value, function, subcellular location, and UniProt hyperlink
- Pathway analysis shows KEGG pathways visualized through DAVID for proteins differentially regulated in an experiment
- FDR shows the number of peptides and proteins detected along with the peptide and protein FDR rate
- Endogenous references has the reference ID number, sequence, precursor m/z, and MS1 intensity for all references
