Spectrum Mill MS/MS Search

Table of Contents

• Introduction
• What is a Fragment-Ion Tag?
• Search Mode
• Definitions
• Comparison of search modes
• Search mode translator across Spectrum Mill versions
• Homology Mode
• Precursor Mass Shift
• Ranking / Scoring of Results
• Scoring Scheme
• Guidelines for Score Interpretation
• Fragmentation and Sequence Content
  
• Discriminant Scoring
• Instrument / Fragment-Ion Types
• Selecting Thermo Fisher Scientific Instruments
• Multiply-Charged Ions
• Immonium Ions / Compositional Marker Ions
• Minimum Scored Peak Intensity
• Mass Tolerances
• Batch Size
• Reversed Database Search
• To Use the MS/MS Search Form
  • Search
  • Data Directories
  • Search Parameters
  • Modifications
  • Search Criteria
  • Matching Tolerances
  • Spectral Quality Filtering
  • Search Mode
  • Data Files  

Introduction

The MS/MS Search module in Spectrum Mill automates the search of processed MS/MS spectra against protein or DNA databases. The MS/MS Search algorithm uses intelligent parallelization to provide extremely fast searches. It can operate in identity mode to find unmodified peptides or in variable modifications or homology modes to look for mutations, post-translational modifications, and chemical modifications.

As you process data with Spectrum Mill, you may iterate through multiple rounds of database search and results validation, with the goal of identifying as many spectra as possible. Spectrum Mill provides a means to segregate search results that contain a valid interpretation of an MS/MS spectrum from those that do not. Spectra that do not have validated matches can then be subjected to subsequent rounds of searches (against larger databases or in variable mode, for example). Spectrum Mill retains a cumulative list of validated matches that you can summarize at any time in the process.

What is a Fragment-Ion Tag?

A Fragment-Ion Tag can be obtained from an MS/MS spectrum and consists of three attributes:

A peptide precursor-ion mass: Pm. Masses of all sequence related fragment-ions from the peptide: Fi, Fj, ... The fragment ions need not all be of the same ion type. Supported fragment-ion types are described below. Masses of composition ions which indicate the presence of particular amino acids in the peptide: Ci, Cj, ... This information can be from immonium and related low mass ions or high mass ions representing side-chain losses from the precursor ion.

Search Mode

Definitions

The following modes are available for MS/MS Search:

• Identity: The MS/MS spectra are compared to unmodified sequences from the database.
• Variable modifications: In addition to searching for identical matches, the search includes the variable modifications you have selected. These may be post-translational modifications or other variable modifications. Since they are considered as possible modifications, the search does not force universal application of variable modifications. Peptides can have more than one variable modification, within the limits of the user-specified Precursor mass shift. The search does not currently support multiple types of variable modifications to the same type of  amino acid. For example, you cannot select both guanidination and carbamylation of lysine as variable modifications within the same search.
• Homology - All mutations: In addition to searching for identical matches, the search includes matches that are consistent with a single amino acid substitution. The substitution does not need to be one that would result from a point mutation. If you have selected variable modifications for an amino acid, they are also considered.
• Homology - Single base pair mutations:  In addition to searching for identical matches, the search includes matches that are consistent with a single amino acid substitution that would result from a point mutation (single base substitution within a codon). If you have selected variable modifications for an amino acid, they are also considered.

To search for an unknown or unexpected modification, select one of the homology modes described above, then click Unassigned single mass gap. This search looks for an unexpected modification (a mass gap). It is a way of doing an "error tolerant database search." Note that you cannot simultaneously search variable modifications in this mode. This type of search is best done when searching previous non-validated hits.

Tips:

Searches run faster and generate fewer false positives when fewer modifications are considered.

Your system administrator can define new mutation/substitution matrices. Do not attempt to modify the existing homology modes.

Comparison of search modes

*Exact matches take into account fixed modifications, which are applied universally to their respective amino acids. Exact matches also take into account any mix modifications that are applied during the search cycle.

**You must select the variable modifications you wish to search (Choose... button)

Search mode translator across Spectrum Mill versions

Homology Mode

In order to match one's data to a sequence in the database that is not identical to the peptide used to generate the MS/MS spectrum, MS/MS Search must be used in homology mode. This enables matching for peptides with a mutation, cross-species substitution, sequence polymorphism, or error in the database. Homology mode works based on three concepts:

1. Allow precursor mass to be shifted from the precursor mass of sequences in the database.
2. Consider each ion independently rather than examining relationships between ions.
3. When a precursor ion undergoes fragmentation, at least two pieces are formed (a fragment-ion and a neutral). While only the mass of the ionized fragment is measured, the mass of the neutral piece is easily calculated as precursor mass - fragment ion mass. If the peptide matches a database sequence exactly, then the masses of BOTH the fragment-ion and the neutral will match. If there is a single sequence difference, then the mass of either the fragment-ion or the neutral will match, but NOT BOTH. If there are two sequence differences, fragmentation at any sites located BETWEEN the two mismatched sites will result in NEITHER the fragment-ion nor the neutral matching.

Matching sequences are filtered through a mutation matrix to try to find a single amino acid (AA) substitution that would transform the calculated mass of the database sequence to the experimentally determined mass. The output displays the necessary substitution and the corresponding sequence consistent with the experimental peptide mass data (not the sequence present in the database).

Precursor Mass Shift

MS/MS Search only considers database sequences with calculated precursor masses which pass through a precursor mass filter. In Identity Mode, the filter is determined by the specified precursor mass +/- the precursor mass tolerance. In Homology mode this is determined by the specified precursor mass and the precursor mass shift. You should NOT attempt to accomplish this by using a wider precursor mass tolerance. Use a precursor mass tolerance consistent with the accuracy to which the precursor mass is measured. The default value of +/- 130 allows for the largest possible precursor mass shift associated with a mutation among the 20 standard amino acids and phosphorylation. All database sequences with a calculated precursor mass + / - 130 Da of the specified precursor mass would thus be considered. This means a large increase in the number of sequences considered, and hence increases the potential for false-positives. The +/= and -/= features allow you to specify an anticipated precursor mass shift value and reduce the number of sequences considered in a search. For example, suppose you expect a phosphorylated peptide; specifying a precursor mass shift of +/= 80 would allow matches to database sequences that exactly match the precursor mass or database sequences that would match the specified precursor mass if 80 Da were added.

The default precursor mass shift range for the Variable modifications search mode is -18 to 177. You can change this setting to encompass the number and type of modifications you expect for your sample.

To summarize, the shift can be set in four different forms, all of which show only homologous matches, thus excluding identity mode matches:

• +/- (wide range) - allows matching of a query spectrum to all library spectra spanning the range of the Precursor mass shift
• =/+/- allows a query spectrum to match a library spectrum only if the query spectrum's precursor MH⁺ is shifted either higher or lower by the specified mass. (The program automatically takes into account precursor charge.)
• +/= (specified shift up) - allows a query spectrum to match a library spectrum only if the query spectrum's precursor MH⁺ is shifted higher by the specified mass. (The program automatically takes into account precursor charge.)
• -/= (specified shift down) - allows a query spectrum to match a library spectrum only if the query spectrum's precursor MH⁺ is shifted lower by the specified mass. (The program automatically takes into account precursor charge.)

Note that the +/- will compare many more spectra so it will take longer to run, and the run time will be proportional to the magnitude of the Precursor mass shift.

Ranking / Scoring of Results

The explanations below are for a simple score calculation. With version B.04.00 and later, you also have the option to select Discriminant Scoring.  See Discriminant scoring to learn more about the "figures of merit" that comprise the discriminant score and search mode to learn about the discriminant scoring options from which you can choose.

Scoring Scheme

Following peak detection, the MS/MS Search algorithm attempts to match every ion present in an MS/MS spectrum to an ion type consistent with fragmentation of a peptide sequence from a database. The scoring system is information-content oriented and based on the following general principles:

• Peak Intensity - If a peak is "real" and explainable, intensity doesn’t matter. Very intense unexplained peaks suggest poor interpretation.
• Fragment Ion Types - Secondary ion types have redundant information content.
• Peptide Fragmentation - Sequence completeness depends on backbone cleavages (e.g., b or y ions).
• Peptide Length - Longer peptides yield more ions and thus the spectra contain more information.
• Protein Identity - One good peptide MS/MS is better than three mediocre ones.
• Proton Mobility - This scoring refinement for ion trap MS/MS data is based on the likelihood of fragmentation at a particular location. When you mark the check box for proton mobility scoring in MS/MS Search, the software takes into account relative proton mobility based on peptide charge state and amino acid composition. It also considers the position of an amino acid in a peptide and the peptide length. For details, see:

Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O'Hair, R. A. J.; Speed, T. P.; Simpson, R. J.; "Mining a Tandem Mass Spectrometry Database To Determine the Trends and Global Factors Influencing Peptide Fragmentation;" Anal. Chem.; 2003; 75(22); 6251-6264. DOI: 10.1021/ac034616t

Do not use proton mobility scoring when peptides are modified in a way that significantly alters the way the peptide fragments.  You can use proton mobility scoring for ¹⁴N/¹⁵N, SILAC, and ¹⁶O/¹⁸O because these isotopic labels do not change the structure of the peptides and thus do not change the way these peptides fragment. However, the iTRAQ labels and the lys-imidazole labels do change the way a peptide fragments and thus proton mobility scoring is not advised for these. It is also not advised for guanidination or phosphorylation.
 

The scoring scheme is intended to facilitate review/filtering of large numbers of spectra (100's - 1000's) that enables segregating valid from false-positive interpretations. Note that probability values associated with the number of proteins/peptides in the database are NOT used. Thus the score for a spectrum against a particular candidate sequence will always be the same, as the information content of the spectrum is database-independent.

MS/MS Search scoring has four particular attributes. Only the first two are used for post-search review/filtering purposes within the Protein/Peptide Summary portion of Spectrum Mill.

• Score - The software calculates the MS/MS Search score as described in the following bullets and diagram. To see details about the ions that contribute to any given score, click the link under the Filename header in Protein/Peptide Summary.
  • Bonus points for each peak assigned to an allowed fragment ion type for a candidate peptide sequence. Bonus values of particular ion types are instrument-dependent.
    example: (ESI-ION-TRAP b/y: 1, a: 0.25) Marker ions can also contribute to bonus scoring.
  • Penalty points for each unassigned peak. Penalty value is based on peak height, - (peak height / height of tallest peak).
    example: if an unassigned peak is 50% the height of the tallest peak in the peak-detected spectrum, then its penalty value is 0.5, while an unassigned peak that is 10% the height of the tallest peak has a penalty value of only 0.1.

  

• SPI - Scored Peak Intensity - From peaks remaining after peak detection, the percentage of total intensity which is assigned to particular ion types. Scored Peak Intensities lower than 70% suggest a poor interpretation, or presence of fragment ion types unknown to MS/MS Search.
• BCS - Backbone Cleavage Score - Number of amino acids cleaved between either b or y ions.
• Unmatched ions - # of unassigned ions / # of ions remaining after peak detection.

Guidelines for correlating peptide score with the correct interpretation and the extent of peptide fragmentation:

Q-TOF

• Score > 15 Outstanding, well-assigned, thorough fragmentation
• Score > 9 Good
• Score > 5 Mixed Quality
• Score < 5 Generally poor with less fragmentation
• SPI < 60% Generally poor with significant unassigned ion current
  

Ion trap

• Score > 15 Outstanding, well-assigned, thorough fragmentation
• Score > 10 Good
• Score > 5 Mediocre, relatively few fragment ions produced, look for mostly b's or mostly y's.
• Score < 5 Poor, little to no fragmentation
• SPI < 70% Poor, significant unassigned ion current

Fragmentation and Sequence Information Content

The figure below illustrates some of the diversity of quality and information content in MS/MS spectra.

If scores for a single spectrum against multiple candidate sequence are identical, the results are then sorted so that if multiple sequences are matched, more likely sequences are listed higher in the list and sorted on the following basis:

1. In Variable modifications mode and in Homology mode, matches to database sequences identical to their appearance in the database are listed higher.
2. In Variable modifications mode and in Homology mode, matches with known modifications are listed higher.
3. Sequences matching with the least number of unmatched ions are listed higher.
4. Among equivalent matches, the results are sorted in order of increasing precursor mass.
5. Among equivalent matches, the results are sorted in order of increasing index number.

Note that the last two sorts do NOT imply a BETTER ranking, even though one match will be listed higher than another, but are merely intended to provide some organization to the listing and to aid the user in viewing the results.

Discriminant Scoring

• Score
• Delta rank1-rank2 score
• Backbone cleavage score
• Number of complementary fragments
• Maximum matched sequence tag length (b,y,c, or z ions)
• SPI%
• Percentage of matched ions (e.g., 17/25 peaks matched = 68%)
• Standard deviation of fragment mass errors/fragment tolerance
• Charge state

Instrument / Fragment-Ion Types

Selecting an instrument triggers the configuration of MS/MS Search scoring and peak detection parameters designed particularly for the type and extent of peptide fragmentation observed on that instrument. The particular parameters can be edited or new instruments added by editing the files: msparams_mill/instrument.txt, and millhtml/SM_js/instrument.js

E:\SpectrumMill\msparams_mill\instrument.txt  
E:\SpectrumMill\millhtml\SM_js\instrument.js  

If you add a new instrument type, be sure to set the parameters  in instrument.txt in a way that is appropriate for the data you export from that instrument. For example, if deisotoping is accomplished by  the instrument data system, set bypassDeIsotoping = 1 in instrument.txt to avoid repeating deisotoping in Spectrum Mill.

Examples of supported instrument configurations are shown in the three tables below.  Agilent ESI instruments are described in the first table, while other ESI instruments are described in the second. MALDI instruments are described in the third table. For additional supported configurations and the latest updated settings, see E:\SpectrumMill\msparams_mill\instrument.txt.

Table 1. Examples of supported configurations for Agilent instruments

*N/A = not applicable

Table 2. Examples of ESI configurations

*N/A = not applicable

Table 3. Examples of MALDI configurations

*N/A = not applicable

Selecting Thermo Fisher Scientific Instruments

If you have a Thermo Fisher Scientific Orbitrap or LTQ FT, select your instrument based on where the MS/MS occurs.

• If you have an Orbitrap:
• If MS/MS occurs in the LTQ, select the appropriate linear ion trap instrument and change the Precursor mass tolerance to 0.05 Da (note that the units must be  Daltons).
• If  MS/MS occurs in the Orbitrap, select the appropriate Orbitrap instrument and keep the defaults (in ppm).
• If you have a standard LTQ FT, select the appropriate linear ion trap instrument because the MS/MS occurs in the LTQ. (Only precursor ion scans occur in the FT.) Set the Precursor mass tolerance to 0.05 Da.
• If you have an LTQ FT Ultra, and MS/MS occurs in the FT, then select ESI Orbitrap.

Fragmentation modes and location

• CID - collision-induced dissociation in the LTQ
• ETD - electron transfer dissociation in the LTQ
• HCD - isolate precursor ions in the LTQ, dissociate in the C-trap, scan out the fragment ions and detect in the Orbitrap
• PQD - pulsed-q dissociation - isolate precursor ions and dissociate in the LTQ, but without the low-mass cutoff that you see with CID spectra

Multiply-Charged Ions

When data is of sufficient resolution that charge state can be determined from the isotope distribution, and the software designates MS/MS Search instrument configuration as "fragment charges certain,"  then masses are converted to charge 1 inside MS/MS Search prior to interpretation. However, the charge state is still used to evaluate matching sequences to check that they contain a sufficient number of basic residues to support the charge. Further, in the output, the labels distinguish whether the ion type used inside MS/MS Search was of the converted to charge 1 high res variety (y⁺²) or of the ambiguous low res variety (y++).

Immonium Ions / Compositional Marker Ions

Marker ions represent peaks that indicate amino acid composition, but do not indicate sequence. The table below describes the allowed amino acid composition marker ions. In general, the scores correspond to the rarity of the amino acids as described by the number of codons coding for the amino acids that can produce the ion.

Note that the file msparams_mill\smconfig.xml defines additional marker ions and their scoring for a large number of amino acid modifications. The scoring is invoked when the fixed and variable modifications are selected for the search. System administrators can add custom modifications, along with their marker ions.

For scoring purposes, one can not make a yes/no distinction between marker ions and peaks that are isobaric with marker ions. So for scoring purposes, Spectrum Mill shrinks the intensities of marker ions to 10% of their original intensities. This enables them to be matched when they are isobars, without the intensities causing hit rejection when they are marker ions.

Minimum Scored Peak Intensity

Prior to performing scoring, MS/MS Search first screens the MS/MS spectrum against candidate sequences using a simple filter. This filter is Minimum scored peak intensity. This approaches enhances search speed by allowing candidate sequences to be rapidly and summarily rejected once a sufficient number of spectral peaks are examined and found not to meet the threshold established by this filter.

For ultimate coverage in MS/MS Search, lower the Minimum scored peak intensity. When there are one or more very intense peaks that overwhelm other peaks but cannot be assigned, setting this value to near 0% may improve the number of hits at the expense of longer search times.

Guidelines: Since the matching which occurs before scoring is dependent on this filter, the value should be set in relation to one's expectation of the quality of peak detection, i.e. noise removal and selection of  ¹²C isotope peaks representing fragment ions corresponding to the selected Allowed Fragment-Ion types in the spectrum prior to searching. This parameter has a very significant impact on search speed; the more unmatched peak intensity allowed (lower percentage), the longer the search time. Composition ions are counted as unmatched intensity, but only at 1/10 their actual peak height.

Mass Tolerances

The tolerances on both the precursor ion and fragment ions should be set to be consistent with the mass accuracy of the instrument used to generate the data. For spectra from time-of-flight instruments, it is generally a better idea to use units of ppm or % rather than Da, since mass accuracy is often better at lower mass than at higher mass. 

Batch Size

When you run MS/MS Search, the batch size determines the maximum number of spectra analyzed in one pass through the database. Since all spectra of similar charge states are grouped together before splitting into batches, the last batch for each charge state will likely contain fewer spectra than the maximum batch size.

For maximum search speed, the optimum batch size depends on the size of the database, the type of search (identity, variable modifications, or homology), the number of modifications, and the mass accuracy of the instrument. If the batch size is too large for the complexity of the search, the search may time out and fail to complete. For a complex search, there is no advantage to using a larger batch size because the majority of the search time results from the database matching rather than setting up the batches. The following table provides guidelines for the batch size you should enter into the MS/MS Search form.

With B.04.00 and later, larger batch sizes may be specified without the risk of timeouts. If Maximize CPUs is marked, best performance is with a batch size of 150 or more. The default batch size is now 500. If you have less than 16 Gb of memory and are searching large data sets, use a batch size less than 500.

If you have a Thermo Fisher Scientific Orbitrap or LTQ  FT, or another  instrument that produces spectra with high mass accuracy, follow the guidelines for Agilent Q-TOF. If you have an ion trap or other  instrument that produces spectra with lower mass accuracy, follow the guidelines for Agilent ion trap.

During searches, the Spectrum Mill software dynamically reduces the batch size as the m/z increases. For variable modifications searches and homology searches, the number of possible combinations rises dramatically with increasing m/z; by dynamically reducing the batch size, the software reduces memory usage.

Reversed Database Search

A reversed database search helps to rule out false positives and allows the software to calculate a false discovery rate. If you obtain similar scores for both forward and reversed searches, there is a higher likelihood of an incorrect assignment.

For a reversed database search, the Spectrum Mill software reverses only the internal portion of the peptide sequences in the database rather than reversing the complete database itself. For example, the peptide:

SAMPLER

is reversed to

SELPMAR

rather than

RELPMAS.

All of these internally-reversed sequences from the database are compared to the MS/MS spectrum and the one that returns the highest score is saved as the reversed database hit. The reversed database hit is not always the reverse of the peptide that matched in the forward search, because a different reversed hit may score higher. That is, all of the possible reversed hits are considered as potential matches for the experimental spectrum.

For spectra with high mass accuracy data, such as Agilent Q-TOF, many sequences will not have a reversed hit.

To Use the MS/MS Search Form

The following topics describe options available on the MS/MS Search form.  In general, you should retain the default settings, except for the options highlighted in red text on the form.

See the rest of this document for more details regarding MS/MS Search.

Search

• Start Search - Click to place the task in the queue for execution.  The program determines the order in which it will execute the task to do an MS/MS search based on the time the task entered the queue, its capacity to execute tasks in parallel, and dependencies. Click this button after you have either loaded the desired parameter file or manually set the parameters. The name of the current parameter file appears in red at the top of the form. Once you have saved a parameter file from this form, you may start the search from a workflow rather than manually with the Start Search button.
• Save As - Click to save current search settings in a parameter file. 
• Load - Click to load a parameter file that contains settings for MS/MS Search. For default values, select a parameter file from the Defaults folder.
• Remove all prior MS/MS Search results - Mark this check box to remove prior MS/MS search results for this dataset.  This will also remove Spectrum Summary results.
• Maximize CPUs - Mark this check box if you want this search to take advantage of all available CPUs (as opposed to using only a single CPU so that the other CPUs are available for other processes/users). Best performance is obtained with a Batch size of 150 or more. If you mark this check box for a workflow, the request queue will show two requests -- the initial one to create the batch (of files) and the other to show the progress and search results. 

Data Directories

• Click the Select ... button to select a data directory or data directories.  See Selecting Data Directories.

Search Parameters

• Validation filter:  Use this to search spectra having only a particular validation setting. See Peptide Validation.
• Batch size - See Batch Size.
• Search previous hits: Mark this check box to search valid hits saved from a previous search. See Saving Hits. If you have not yet saved the valid results using the Tool Belt form, this search page does it for you. If you mark the check box for Remove all prior MS/MS Search results, then the software ignores the setting for Search previous hits (since you will have removed all previous results).
• Max. reported hits:  Set to the maximum number of hits you want for each search.
• Database:  Select a database.  See Databases.
• Species:  Choose a species if you want to narrow the search possibilities and to accelerate searches.  Please see the list of species definitions that ship with the software, as some definitions do not encompass all possible members.  Retain the default of All to search the entire database.  Be aware that because of inconsistencies in the way species information is organized in different databases, Spectrum Mill cannot read about 10% of the species information in NCBInr, and cannot read any of the species information in trEMBL.  See Species Filtering.
• Digest:  Select the enzyme used for the proteolytic digestion.  See Enzyme Specificity / Missed Cleavages. If you select No enzyme, then the software ignores Maximum # missed cleavages.
• Maximum # missed cleavages:  Set the maximum number of missed enzymatic cleavages.  For homology searches (or searches with many variable amino acid modifications) against a full database, reduce the number from the default of 2 to 1. See Enzyme Specificity / Missed Cleavages.
>

Modifications

• Click the Choose... button to select modifications appropriate for your sample.  See Choosing Modifications.

Search Criteria

The next several topics describe options available in the Search Criteria section of the MS/MS Search form.

Matching Tolerances

• Disable match filtering (SPI, STL, S/N filter) - Mark this check box if you wish to compare results with those from other database search engines. CAUTION: Because this mode disables signal-to-noise and spectral quality filtering, some of the spectra you submit for the search will be poorer quality and you will generate significantly more false positives! See Disable quality filtering mode/disable match filtering modes. Note that the check box for Disable match filtering is available only if it is configured in SMglobals.js. See the server administration help for details.
• Minimum scored peak intensity:  See Minimum Scored Peak Intensity.
• Instrument:  Select the instrument used for the analyses.  See Instrument / Fragment-Ion Types. For Thermo Fisher Scientific Orbitrap and LTQ FT, see Selecting Thermo Fisher Scientific Instruments and Fragmentation Mode below.
  
• Masses are:  See Mass Type.
• Precursor mass tolerance:  See Mass Tolerances.
• Product mass tolerance:  See Mass Tolerances.
• Maximum ambiguous precursor charge:  Select the maximum charge state that you want MS/MS Search to use when it encounters an extracted spectrum for which the charge state is unknown (an extracted file that ends in *.0.pkl). While the default of +3 is optimized for CID spectra and trypsin digestion, it may be advantageous to select higher charge states for ETD spectra or when the proteolytic enzyme produces longer peptides (for example, the enzyme is LysC or GluC).

Spectral Quality Filtering

Search Mode

The latter three options apply only in certain homology modes.

• Calculate reversed database scores:  Mark this check box if you wish to perform a database search against peptide sequences in their forward and internally reversed directions. If you obtain similar scores for both searches, there is a higher likelihood of an incorrect assignment. Such a search helps to rule out false positives. See Reversed Database Search. You must mark this check box if you want to calculate a false discovery rate (FDR), either in Autovalidation or using the Tool Belt utility.
• Protein mobility scoring: Mark this check box if you have ion trap spectra and you wish to have database search scores take into account the likelihood of fragmentation at a particular site, based upon relative proton mobility and other factors. See Ranking/Scoring of Results. Do not mark this check box if you have peptides that are modified by iTRAQ, lysine mass tagging, guanidination, or phosphorylation, because these modifications change the expected fragmentation pattern.
• Dynamic peak thresholding: Mark this check box if you wish to use a scoring enhancement that enables identification of more low-abundance and short-chain peptides. For each extracted spectrum, the software calculates the search scores as the number of spectral peaks varies from n=4 up to the maximum set by the variable peakLimitCount in instrument.txt. It then displays the best score from the set.
• Discriminant scoring: The default selection is "Off", which disables the creation of the files that are necessary for use of the Autothresholds – discriminant strategy in autovalidation. The second selection is "Score only." It replaces the "Disable" field from the prior release and is here for backwards compatibility. However, "Score only" negates the purpose of discriminant scoring, which is to use a combination of other factors in the score. If you choose one of the selections below, discriminant scoring is enabled and uses the calculated set of coefficients for the Agilent instrument selected, or for an instrument that you choose. You can create a set of coefficients for an instrument of your choosing with the Tool Belt utility. After you do this, the name of the set of coefficients appears in the selection list.
• Agilent QTOF standard - Discriminant scoring will use this set of already calculated coefficients for the Agilent QTOF.
  
• Agilent Ion Trap CID standard - Discriminant scoring will use this set of already calculated coefficients for the Agilent ion trap CID fragmentation mode.
  
• Agilent Ion Trap ETD standard - Discriminant scoring will use this set of already calculated coefficients for the Agilent ion trap ETD fragmentation mode.
  
• Search mode:  Select a search mode. See Search Mode. If you select the variable modifications or one of the homology modes, the software displays one or more of the following options:
• Precursor mass shift or Precursor mass shift range:  The default setting depends on which homology mode you use. See Precursor Mass Shift. In the Variable modifications search mode, set a number that is appropriate for the types and numbers of modification(s) that you are searching. Larger values may produce longer searches and increase the possibility of false positives.
• Single AA substitution / Unassigned single mass gap - In one of the homology modes, if you wish to search for a single amino acid substitution, select Single AA substitution. If you wish to instead search for an unknown modification, select Unassigned single mass gap.

Data Files

• Fragmentation Mode - Select one of the modes below for an ion trap Instrument.  These selections take the place of the ion trap "MIX" Instrument selections found in versions previous to B.04.00.  For Agilent Q-TOF and other instruments that only acquire CID, always specify All.
  
• CID - collision-induced dissociation via resonance excitation in the ion trap
• ETD - electron transfer dissociation in the ion trap
• ETHCD - electron transfer dissociation in the ion trap, transfer all ions to the HCD collision cell, dissociate via beam-type collision induced dissociation.
• HCD - isolate precursor ions in the ion trap or with Q1, dissociate in the HCD collision cell via beam-type collision induced dissociation, scan out the fragment ions and detect in the Orbitrap
• PQD - pulsed-q dissociation - isolate precursor ions and dissociate in the ion trap, but without the low-mass cutoff that you see with CID spectra
• Spectrum Files - Modify this list if you want to process only a subset of the files in the data directory.  Wildcards (*) are supported.