Solutions > Mascot PMF overview

Mascot for Peptide Mass Fingerprinting (PMF)

Mascot PMF is a high-throughput system for identifying proteins from Peptide Mass Fingerprinting (PMF) data. It is built on three products: Mascot Distiller (raw data processing), Mascot Server (high-throughput identification) and Mascot Daemon (automation).

Peptide mass fingerprinting can only be used with a pure protein or a very simple mixture, so the starting point will often be a spot off a 2D gel. The protein is digested with an enzyme of high specificity, usually trypsin, but any specific enzyme can be used. The resulting mixture of peptides is analysed by mass spectrometry, yielding an MS scan containing molecular mass values. The protein is identified by searching the MS spectrum against a database of protein sequences.

In addition to PMF, Mascot supports DDA and DIA analysis, where the peptides are subjected to gas phase fragmentation (also known as MS/MS or tandem MS).

Why should I use Mascot for PMF?

Mascot PMF is ideal for high-throughput identification of proteins from PMF data because of its flexibility.

Use any sequence database, from a single proteome to the entire UniProt database (hundreds of millions of sequences).
Use any variable modifications from Unimod (~1500 mods) or add your own.
Use any enzyme or enzyme mixture.
High-throughput client-server architecture – any number of client programs can use the same protein identification server.

Mascot is ideal for PMF because of its reliability.

Universal probabilistic scoring model – works with any instrument.
Universal peak detection – works with any instrument.
Protein identifications are inherently reliable – Mascot calculates a statistical significance for each hit.

What do I need to get started?

PMF support is built in to all versions of Mascot Server. You are welcome to use the free service at matrixscience.com for trial purposes.

You will need to convert your raw data into peak lists in Mascot Generic Format (MGF) format. Many software packages and free tools are available:

Mascot Distiller is a vendor independent application for processing raw data.
Alternatively, use an instrument data system (for example, Bruker BioTools, Shimadzu MALDI Solutions Data Acquisition).
Alternatively, use a free tool like ProteoWizard msConvert.

If you need a new licence or wish to evaluate Mascot Server on your own hardware, please contact us or get a quote.

Which instruments are supported?

Mascot PMF is available for all Agilent, Bruker, SCIEX, Shimadzu, Thermo and Waters instruments commonly used in proteomics.

Mascot Distiller has been designed to work with raw data from any instrument. The data file could contain a single, low resolution MS scan or it could be a complex, high resolution, DDA run from a hybrid instrument. All Distiller parameters can be optimised to a specific workflow.

Mascot also supports DDA analysis and DIA analysis.

What is the Mascot PMF workflow?

The first step is to generate a peak list from the raw data. We recommend Mascot Distiller, but you can also use your instrument data system.

A PMF peak list is a set of molecular mass values, which are searched against a database of protein sequences. For each entry in the protein database, the search engine simulates the known cleavage specificity of the enzyme, calculates the masses of the predicted peptides, and compares the set of calculated mass values with the set of experimental mass values. Mascot uses probabilistic scoring to identify the entry in the database that gives the best match. At the end, a report is generated.

In a peptide mass fingerprint, it is the mass values of the peaks that matter most. The peak area or intensity values are a function of peptide basicity, length, and several other physical and chemical parameters. There is no particular reason to assume that a big peak is interesting and a small peak is less interesting. The main use of intensity information is to distinguish signal from noise.

Mascot Daemon automates all steps of preprocessing and database searching.

What kind of PC hardware does Mascot PMF require?

Peptide mass fingerprinting is not computationally intensive. Any recent PC with at least 8 processor cores and 8GB of RAM is suitable.

If you plan to run Mascot Server as a high-throughput system, we recommend a Medium licence with at least 16 processor cores and 16GB of RAM. This allows multiple searches to run concurrently.

You can run Mascot Server and Mascot Distiller on the same PC or different PCs. Mascot Server and Mascot Distiller run on the CPU and do not require a GPU.