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Articles tagged: peak picking
Mascot workflow for LC-MS/MS data
Data analysis in mass spectrometry proteomics is complex and, nowadays, almost entirely software driven. Processing a raw file, peptide identification by database searching, protein inference and protein quantitation all have many steps and built-in assumptions, not to mention a huge number of parameters. Software continues to evolve as does best practice. Whether you are new to mass spectrometry proteomics or [...]
Peak picking intact crosslink spectra with Mascot Distiller
There are several important factors to take into account when carrying out peakpicking for an intact crosslinked dataset. You’ll typically be dealing with higher charge state precursors to handle the larger masses of the linked peptides, and the MS/MS spectra are inherently complex, chimeric spectra with fragments from the alpha and beta peptides. To look at the effects of adjusting [...]
Default or prof_prof? Peak picking Thermo .RAW data with Mascot Distiller
We supply a number of processing options files for each of the main vendor raw file types with Mascot Distiller. These .opt files are designed as a reasonable starting point for peak picking your own data – but to get the very best you’ll need to tweak the parameters on a typical raw file from your instruments and then use [...]
The Third Column: MS/MS fragment ion decharging in Mascot Server 2.7
In the Mascot Generic (mgf) peak list format, MS/MS fragment ions are typically defined as pairs of values. The first value is the m/z of the fragment ion peak and the second the intensity of the peak. However, a third value can also be supplied – the charge state of the fragment ion. In Mascot 2.6 or earlier, the fragment [...]
Mascot Distiller 2.7: Farewell to re-gridding
We recently released Mascot Distiller 2.7. The main new feature of this release is a change to how peak detection works on raw profile datasets that have been saved as sparse, or compressed data, with runs of zero values dropped. Common examples of this are Thermo Orbitrap and Sciex Analyst datasets saved as profile data. For these types of data, [...]
How many of you are there in there? Processing and searching chimeric MS/MS spectra with Mascot Distiller and Mascot Server
In the typical shotgun proteomics experiment, the assumption is that each MS/MS spectrum is derived from a single precursor selected by the Mass Spectrometer for fragmentation. However, in practice, near isobaric precursors can co-elute and undergo co-fragmentation resulting in chimeric MS/MS spectra containing fragments from multiple different precursor peptides. With high resolution data, it is possible for these overlapping isotope [...]
Some peaks are more equal than others
When you look at the details of a peptide match in the Mascot Peptide View report, only a small number of the peaks may be labelled in the spectrum graphic and highlighted in the table of fragment masses. We often got challenged about this: "Why haven’t you labelled these other peaks that clearly match?". So, in Mascot 2.3, we added [...]
Peak list arcana*
This article addresses some aspects of how the information in a peak list is used by Mascot, what is not used, and how the peak list is processed prior to a search. These things are all in the manual, but can be difficult to find, short of reading it from cover to cover. Fragment charge is not used in the [...]
Current challenges in quantitative proteomics
"Current challenges in software solutions for mass spectrometry-based quantitative proteomics" is a recent paper in Amino Acids by a group of expert authors that describes ten areas of particular difficulty in data processing for quantitation. Full text is available online at Springer Link. I would argue that Mascot Distiller meets almost all of these challenges. Obviously, I have to declare [...]