The PEAKS team was extremely busy in the past several weeks and finally today we are able to put PEAKS 7 beta in our invited testers' hands.
The biggest new feature in PEAKS 7 is the new label free quantification algorithm, which will replace the old algorithm in the optional quantification module. Our HUPO 2013 poster, PEAKS - A Software Tool for Shotgun Label Free Proteomics with High Sensitivity and High Accuracy, showed some preliminary results. To help our user navigating through the data and results for manual inspection, we have re-engineered the 2D heatmap and 3D view to provide a smooth, Google Map-like experience.
PEAKS is already the best commercial de novo sequencing solution. In PEAKS 7, we make it even better by tackling two fronts: improving accuracy and providing result validation/filtration. This poster shows that when the peptides are fragmented using different fragmentation methods the accuracy of de novo sequencing can be significantly improved. PEAKS 7 also includes tools and guidelines for de novo result filtering and validation, described in this poster.
There are many more improvements in PEAKS 7, we will unveil them at release, shall we?
We may expand our tester base slightly in the next few weeks depending on the stability of the beta build. If you are willing to get early access to PEAKS 7, please comment on the post or send email to peaksbeta@bioinfor.com. Although we cannot guarantee your early access, we will make sure that you get notified as soon as PEAKS 7 released.
Wednesday, October 16, 2013
Wednesday, October 2, 2013
Local confidence score and de novo tags
PEAKS de novo sequencing not only produces accurate de novo sequences from the spectrum, it also provides confidence score at amino acid level.
In the de novo table, when you hover the mouse cursor on a de novo sequence, the following window will show up to display the local confidence score for each amino acid.
The sequence is color coded. Red represents a very high confidence (greater than 90%), purple represents a high confidence (80 to 90%), blue represents a medium confidence (60 to 80%), and black represents low confidence (less than 60%).
PEAKS also provide a slider to change the low confident amino acid to mass tag. We call the remaining consecutive high confident amino acids de novo tag.
Those de novo tags may then be used in BLAST.
In the de novo table, when you hover the mouse cursor on a de novo sequence, the following window will show up to display the local confidence score for each amino acid.
The sequence is color coded. Red represents a very high confidence (greater than 90%), purple represents a high confidence (80 to 90%), blue represents a medium confidence (60 to 80%), and black represents low confidence (less than 60%).
PEAKS also provide a slider to change the low confident amino acid to mass tag. We call the remaining consecutive high confident amino acids de novo tag.
Those de novo tags may then be used in BLAST.
Friday, September 6, 2013
PEAKS at HUPO 2013 - Yokohama, Japan
The PEAKS team is getting ready to attend the HUPO 12th Annual World Congress. The conference will start on September 14th. This year's theme is "The Evolution of Technology in Proteomics".
We will be exhibiting at booth #11. On our booth, you can get a hand on experience on our current version PEAKS 6 to see how it may help in your research. Additionally, you can take a sneak peek at our upcoming PEAKS 7, which will be released in November. We will also have the help from our Japanese distributor at our booth to better serve the local researchers.
Please drop by at booth #11 if you are going to Yokohama for HUPO congress. If you are busy and could not make it there this time, you can always get the latest information about PEAKS from our website.
We are looking forward to seeing you in Japan.
Friday, August 30, 2013
CHAMPS Antibody Sequencing Workflow
A couple of months ago, we have announced our CHAMPS antibody sequencing service. With the FREE "blind trials" we offered during the promotional period, we have received quite a few dataset from several users. The responses to the results we provided are remarkable.
We require the sample to be reduced with DTT, alkylated with iodoacetamide. Glycans need to be removed and heavy/light chains must be separated. Each chain then will be digested with six enzymes: AspN, chymotrypsin, GluC, LysC, pepsin and trypsin. MS/MS spectra is required to be acquired using LTQ-Orbitrap at high resolution with HCD fragmentation. In total, we require six LCMS runs per chain.
The data analysis starts off from PEAKS de novo sequencing. A list of high quality de novo sequences will be generated along with the positional confidence score for each amino acid. Then an in-house developed program will be used to assemble the de novo peptides into much longer sequences, protein contigs. In our experiments, the majority of assembled contigs had a length of 60~120 residues.
We blast the protein contigs in NCBI nr database to assemble the antibody template. We select a protein hit corresponding to the constant region and select the closest protein hit corresponding to the variable region. All the contigs will then be mapped to the template to get the first draft of the antibody sequence. In principle, we trust contigs in variable region and the template in constant region.
The draft sequence will be refined iteratively using PEAKS SPIDER homology search. During each iteration, we will examine insertion/deletion/mutation reported by SPIDER, residues with low peptide coverage, residues at the protein n-terminus and compare the sequence mass with protein intact mass, if available.
*Some content in this post is extracted from the ASMS 2013 poster "Whole Protein de novo Sequencing from MS/MS". You can find a web version of the poster here.
We require the sample to be reduced with DTT, alkylated with iodoacetamide. Glycans need to be removed and heavy/light chains must be separated. Each chain then will be digested with six enzymes: AspN, chymotrypsin, GluC, LysC, pepsin and trypsin. MS/MS spectra is required to be acquired using LTQ-Orbitrap at high resolution with HCD fragmentation. In total, we require six LCMS runs per chain.
The data analysis starts off from PEAKS de novo sequencing. A list of high quality de novo sequences will be generated along with the positional confidence score for each amino acid. Then an in-house developed program will be used to assemble the de novo peptides into much longer sequences, protein contigs. In our experiments, the majority of assembled contigs had a length of 60~120 residues.
We blast the protein contigs in NCBI nr database to assemble the antibody template. We select a protein hit corresponding to the constant region and select the closest protein hit corresponding to the variable region. All the contigs will then be mapped to the template to get the first draft of the antibody sequence. In principle, we trust contigs in variable region and the template in constant region.
The draft sequence will be refined iteratively using PEAKS SPIDER homology search. During each iteration, we will examine insertion/deletion/mutation reported by SPIDER, residues with low peptide coverage, residues at the protein n-terminus and compare the sequence mass with protein intact mass, if available.
*Some content in this post is extracted from the ASMS 2013 poster "Whole Protein de novo Sequencing from MS/MS". You can find a web version of the poster here.
Wednesday, August 21, 2013
De Novo Assisted PTM "Blind Search" - PEAKS PTM
In PEAKS 6, we have introduced a new algorithm for PTM "blind search", PEAKS PTM. This algorithm can search for modified peptides with all 600+ PTMs in the Unimod database. To use this algorithm, simply select the option in the PEAKS Search dialog.
How is the "blind search" achieved? The secret is de novo sequencing. In usual PTM search algorithm, all possible modification forms of all database peptides satisfying the enzyme digestion rules are tried to match the spectra.
In PEAKS PTM, we only search for PTMs on the peptides when there is a tag match*. The algorithm also takes the PTM rarity into account to reduce the search space and false PTM assignment.
With PEAKS PTM algorithm selected, users only need to specify a very small number of variable PTMs in de novo and PEAKS DB to speed up the search and rely on PEAKS PTM to find other modifications presented in the sample.
*X. Han et al. PeaksPTM: Mass Spectrometry Based Identification of Peptides with Unspecified Modifications. JPR 2011, 10(7): 2930-2936.
How is the "blind search" achieved? The secret is de novo sequencing. In usual PTM search algorithm, all possible modification forms of all database peptides satisfying the enzyme digestion rules are tried to match the spectra.
In PEAKS PTM, we only search for PTMs on the peptides when there is a tag match*. The algorithm also takes the PTM rarity into account to reduce the search space and false PTM assignment.
With PEAKS PTM algorithm selected, users only need to specify a very small number of variable PTMs in de novo and PEAKS DB to speed up the search and rely on PEAKS PTM to find other modifications presented in the sample.
*X. Han et al. PeaksPTM: Mass Spectrometry Based Identification of Peptides with Unspecified Modifications. JPR 2011, 10(7): 2930-2936.
Friday, August 9, 2013
de novo only peptides in PEAKS
One of the unique features in PEAKS is that it provides a list of de novo only peptides. What does it mean?
de novo only peptides are the de novo sequences derived from the spectra that do not a have confident database match.
In PEAKS, de novo only peptides are listed in the de novo only tab in every PEAKS DB, PEAKS PTM, SPIDER and inChorus results. The actual de novo only peptides displayed in the list will be affected by the filters in the summary tab. Let's use PEAKS DB result as an example.
The de novo only peptides are defined in the second row of the filters. The first part, TLC and ALC filters are the same as it is in the de novo result. It tells PEAKS what should be considered as a good de novo sequence. The second part, peptide -10lgP filter tells PEAKS what should not be considered as a confident database match. After the filters are applied, PEAKS will go through all the de novo sequences that passed the TLC and ALC filters. For each of such de novo sequence, PEAKS will look at the corresponding spectrum. If the spectrum does not produce any database matches that have a score higher than the de novo only peptide -10lgP filter, the de novo sequence of the spectrum will be considered as a de novo only peptide.
PEAKS does not stop at just providing a list of de novo only peptides, it also tries to associate them with the proteins. In the protein coverage view below, the gray bars represents the de novo only peptides that share at least 10 consecutive AAs with the protein sequence. This is particularly useful when looking for unexpected PTMs or glycosylation site.
de novo only peptides are the de novo sequences derived from the spectra that do not a have confident database match.
In PEAKS, de novo only peptides are listed in the de novo only tab in every PEAKS DB, PEAKS PTM, SPIDER and inChorus results. The actual de novo only peptides displayed in the list will be affected by the filters in the summary tab. Let's use PEAKS DB result as an example.
The de novo only peptides are defined in the second row of the filters. The first part, TLC and ALC filters are the same as it is in the de novo result. It tells PEAKS what should be considered as a good de novo sequence. The second part, peptide -10lgP filter tells PEAKS what should not be considered as a confident database match. After the filters are applied, PEAKS will go through all the de novo sequences that passed the TLC and ALC filters. For each of such de novo sequence, PEAKS will look at the corresponding spectrum. If the spectrum does not produce any database matches that have a score higher than the de novo only peptide -10lgP filter, the de novo sequence of the spectrum will be considered as a de novo only peptide.
PEAKS does not stop at just providing a list of de novo only peptides, it also tries to associate them with the proteins. In the protein coverage view below, the gray bars represents the de novo only peptides that share at least 10 consecutive AAs with the protein sequence. This is particularly useful when looking for unexpected PTMs or glycosylation site.
Monday, July 29, 2013
FDR on combined result from multiple search engines
There are many database search software available for peptide identification. Every software use different scoring functions, thus give them complementary abilities in assigning different spectra from the same MS/MS dataset. This makes combining multiple search engine results a popular methods among researchers.
The tricky part when dealing with the combined results is how to control the quality. In PEAKS 6, we introduced a very easy to use filter in the inChorus result to help resolving this issue. To use this feature, 'Search decoy database from PEAKS' option must be selected for individual search engines when performing inChorus search.
In the 'inChorus' summary view, there is a 'Edit filters' button to control the FDR at the PSM level.
Click the button, a dialog with filter details will be displayed.
The easiest way is to select the target inChorus FDR and PEAKS will automatically determine the appropriate score threshold for each individual search engine so that the combined result has the FDR the user specified. PEAKS also gives users the flexibility to select the score threshold for individual engines manually. When the filters are applied, the overall FDR of the combined result will be calculated.
The tricky part when dealing with the combined results is how to control the quality. In PEAKS 6, we introduced a very easy to use filter in the inChorus result to help resolving this issue. To use this feature, 'Search decoy database from PEAKS' option must be selected for individual search engines when performing inChorus search.
In the 'inChorus' summary view, there is a 'Edit filters' button to control the FDR at the PSM level.
Click the button, a dialog with filter details will be displayed.
The easiest way is to select the target inChorus FDR and PEAKS will automatically determine the appropriate score threshold for each individual search engine so that the combined result has the FDR the user specified. PEAKS also gives users the flexibility to select the score threshold for individual engines manually. When the filters are applied, the overall FDR of the combined result will be calculated.
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