Protein Metrics Preview™ quickly samples MS data to measure mass errors, digestion specificity, and modifications in preparation for more thorough searches. Preview generates optimized parameters files for use in Protein Metrics Byonic™ searches. These parameters include precursor and fragment mass tolerances, digestion specificity, and allowed types of modifications. Preview runs fast initial searches to recalibrate precursor and fragment masses based on confident identifications. Preview samples the data set, but does not perform an exhaustive search, so it is not a substitute for a full database search program such as Byonic.
Main Screen
Preview contains several sections:
Input Files
The first section is Input Files. To load a sample file, click Select MS/MS data file, navigate to and select the MS file and click Open. Most of the sample data file types supported by PMI applications are supported by Preview. These include: Bruker: *.d, Thermo: *.raw, Sciex: *.wiff and *.wiff2, Agilent: *.d, Shimadzu *.lcd, as well as *.mgf, *.mzML and *.mzXML. Preview also supports PMI *.byspec2 files.
To load a protein database (FASTA) file, click Select Protein database file, navigate to and select the *.fasta or *.fa file and click Open. The two filenames are displayed in the cell at the right:
An example fasta file is common.contaminants.fasta found in the directory: C:\Program Files\ProteinMetrics\PMI-Suite\Tools\Preview\data.
Fixed Modifications
The Parameters section contains the Modification options section on the left:
The supported fixed modifications and their available options are:
• Cysteine fixed
For standard cysteine treatments (+0, +46, +57, +58, and +71), this input is not always necessary. If the user selects “(unknown)”, Preview usually determines the correct cysteine treatment from the data.
• Lysine fixed
• Arginine fixed
• N-terminal fixed
To enter a custom modification, enter the mass delta (starting with + or -) in daltons into the appropriate fixed modification cell.
To remove a modification selection, select the blank. In this default mode, Preview will assume 0 Da for Lysine, Arginine, N-terminal, and C-terminal, and will not test for other possibilities for these modifications.
Preview is good at finding modifications that are consistently found sample-wide (for example, in vitro modifications from sample handling and processing). However, searches may miss post-translational modifications that are found only on a few proteins. Note that Preview does not search for glycopeptides. To enable searches of specific post-translational modifications or glycopeptides, carefully review and adjust the Byonic parameters suggested by the Preview output.
Search options
The Search options section, also in the Parameters section, contains fragmentation, digestion and other search parameters:
The Search options include:
• Cleavage site(s) and side set the point of cleavage and whether to cleave on the C-terminal or N-terminal side. Enter one-letter abbreviations for residues on either side of the cleavage point. (in this example, trypsin, on the C-terminal side of arginine or lysine). For a broader search, use nonspecific cleavage at one or both termini. Nonspecific digestion can vary from negligible to ubiquitous depending upon endogenous peptidases, and missed cleavages vary widely depending upon sample processing conditions.
• Initial search specificity
Fully specific searches are recommended for all digested samples. Nonspecific initial searches may perform better for undigested (peptidomic) samples. In Fully specific searches (the default), both peptide termini must agree with the input digestion cleavages. In N-ragged searches, only the C-terminus must agree, and in C-ragged searches, only the N-terminus must agree. In Semi specific searches, one of the two termini can disagree. In Non specific searches, both termini can disagree.
• Check Phospho enriched to optimize Preview when the sample is composed predominantly of phosphopeptides.
• Check Enable wildcard search to enable searches of the spectra with a wild-card modification. When it is checked, the program performs a blind modification search that tries each integer mass shift from -50 to +150 on any one residue. The mass of the modification will be reported to the accuracy of the precursor mass.
• When Try all charge assignments is checked, the charge assignments in the spectrum file are ignored. The program runs every spectrum using z = +1, +2, +3 for each CID spectrum, and z = +2, +3, +4 for each ETD spectrum. You can run Preview twice, once with this box checked and once without, to test the reliability of the charge assignments.
• The Fragmentation type options are:
CID / HCD represent b and y ions, and ETD / ECD represent c and z ions.
Save/Load Parameters
At the bottom of the Parameters section are three buttons to manage the set of parameters
• Load parameters open a collection of parameters (*.prv file) saved in a prior Preview session.
• Save parameters saves the current parameters as a Preview parameter (*.prv) file.
• Reset parameters sets the current parameters back to the default settings.
Run Preview
Before running Preview, the user has the option to automatically open Byonic and load it with the parameters used in Preview. To set this option, check On completion Launch Byonic
If an output folder has not been set before the Preview run, the user will be prompted to set one. To set an output folder, choose Edit > Preferences:, click Select Folder, navigate to and select the desired folder and click Open.
When all parameters and the output folder are set, click Run to start Preview.
A status window opens to show that the run is in progress:
To cancel the process, click Cancel run. To view details of the process, click Show/Hide system output.