Real-Time Library Search filter - Real-Time Library Search filter - Orbitrap Tribrid Apex Small Molecule Method Editor Help - Orbitrap Apex Small Molecule MS - Orbitrap Tribrid Apex Small Molecule Method Editor Help - Filter types - Real-Time Library Search filter - topic

Use Common Library Settings

(Available when multiple RTLS filters are in the method.)

When enabled, synchronize search settings across filter instances.

When enabled, search parameters will be synchronized across instances of this filter where this checkbox is also enabled. When disabled, this instance of the filter will operate with its own search parameters.

Default: Deselected

Use Common Peak Selection and Threshold Settings

(Available when multiple RTLS filters are in the method.)

When enabled, synchronize RTS settings across filter instances.

When enabled, search parameters will be synchronized across instances of this filter where this checkbox is also enabled. When disabled, this instance of the filter will operate with its own search parameters.

Default: Deselected

Library Search Settings

Spectral Library

Spectral Library file location. The library must be the mzVault format (.db).

Collision Energy Tolerance

Collision energy tolerance is the difference allowed between the experimental value and the value used to acquire the reference spectra from the library.

Default: 15

Similarity Search

Performs a similarity search instead of an identify search.

A similarity search will not filter the spectral library by precursor m/z, so every experimental spectrum will be compared against the library content without regard to precursor m/z.

Default: Deselected

Precursor Search Tolerance (ppm)

Specify the precursor search tolerance (in ppm).

Default: 10

Use Fragment Ion Index

(Available when Similarity Search is selected.)

When enabled, build and use a fragment ion index for the search.

Fragment ion index search is helpful for finding matches in your data more efficiently. Enabling this component may use more computer memory (RAM), depending on the settings you choose. This feature is especially useful for broad searches or experiments with multiple precursor search on targeted scans (like tMS²).

When this option is activated, the system looks at the top "N" peaks from your experimental data and checks it against a library. Candidates with matching fragments in the fragment ion index are accumulated. It will then filter out the library candidates that are not similar enough. You can adjust the "Minimum Library Fragment Ion Intensity Contribution" to control how strict this filtering is. The system looks at the remaining candidates and checks if they have enough matched peaks to be considered valid. Remaining candidates with matched ion fragments >= to the "Minimum Matched Peaks" are then scored with the full cosine calculation. Increasing this value will reduce the number of comparisons needed.

Default: Deselected

Minimum Matched Peaks

(Available when Use Fragment Ion Index is selected.)

This value controls the minimum number of peaks required to match between a library spectrum and the experimental spectrum to be considered a valid comparison. Note that the library peaks must also pass the intensity threshold set in the "Minimum Library Fragment Ion Intensity Contribution" setting.

Range: 1–100; default: 4

Minimum Library Fragment ion Intensity Contribution

(Available when Use Fragment Ion Index is selected.)

Peaks with an intensity contribution above the threshold indicated are added to the candidate list for fragment ion indexing. The value specified in the "Minimum Matched Peaks" option will be impacted by the threshold specified here, as the matched peaks are counted and accumulated from the content of the fragment ion index.

This value controls the minimum fractional intensity (relative to the sum signal of the spectrum) required for a library peak to be considered relevant for indexing.

The optimal value here will depend on the nature of the library content. Noisy experimental library spectra may benefit from lower thresholds (0.001–0.02) while synthetic or predicted spectra may benefit from higher thresholds (0.01–0.05).

Range: 0.00–1.00 ; default: 0.05

Number of Experimental Peaks Considered

(Available when Use Fragment Ion Index is selected.)

The number of experimental peaks considered for candidate accumulation during fragment ion index query. For each spectrum, we will query into fragment ion index the top-N intensity peaks, where N is defined here.

Range: 1–500; default: 100

Maximum Search Time (ms)

The maximum time (in milliseconds) allotted for the search to complete.

If the search takes longer than this time, the search stops and returns a failing result. Searches may complete before reaching this time and will continue onto the next acquisition immediately.

Range: 1-2,000; Default: 150

Use Retention Time Filter

Filter library search candidate spectra by the current retention time of the run.

Enabling Retention Time filter will allow you to define the tolerance value, applied as RT ± Tolerance. The unit of time is minutes and must match with your spectral library.

Default: Deselected

Retention Time Tolerance

(Available when Use Retention Time Filter is selected.)

The tolerance with which to allow query candidates for spectral library search, in minutes.

Range: 0.01–600; default: 1

Isotope Error Correction

When the monoisotopic peak is incorrectly assigned, this will query the special library at each C13 offset.

Reverse Library Search

When enabled, perform reverse library search instead of forward search.

Conducts a reverse search, which searches each library spectrum against the query spectrum. If the query spectrum does not include peaks that are in the library spectrum, the match score is negatively affected, but the presence of additional peaks in the query spectrum has no effect on the score. Use a reverse search if the query spectrum includes peaks from several components or has a lot of background noise.

Default: Deselected

Use Multiple Precursors In Search

When enabled, the product spectrum will be searched against multiple precursors and all passing matches will be returned.

Additional precursors are taken from the isolation window in the MS¹ scan, in cases where this information is available within the same experiment, and each is used as the precursor in a separate search for the product spectrum. For cases where MS¹ data is unavailable within the same experiment, like tMS² acquisition schemes, the precursor search tolerance is set to the width of the isolation window.

Default: Deselected

Peak Selection and Threshold Settings

Use as a Trigger Only

Enable passing all peaks through the filter on success.

When enabled and the search produced a passing result, the filter will return all peaks of the generated spectrum, instead of only returning those peaks that are matched to the library.

This is typically used when wanting to perform additional analysis on an ion that produced an identifiable spectrum, but want to select any ion for further analysis.

Default: Selected

TMT SPS MS3 Mode

Enable filtering for TMT-containing fragments.

Your spectral library must either contain library annotations or properly formatted peptide sequence and modification information to make use of TMT mode.

When enabled, the filter will remove any matching fragment ion that does not contain a free amine (Lysines or N-Terminus) where TMT would exist.

When used in tandem with a SPS-MS3 method, this effectively prioritizes SPS peaks that have a high probability of containing a TMT tag.

Consider Unmatched Peaks Only

(Available when the Use as a Trigger Only option is deselected.)

Only pass unmatched peaks through the filter on success.

When enabled and the search produces a passing result, the filter will return unmatched peaks of the generated spectrum instead of only returning those peaks that are matched to the library.

This is typically used when wanting to perform additional analysis on an ion that produced an identifiable spectrum, but want to select unknown fragments only.

Default: Deselected

Modification Trigger

When enabled, this feature will add an additional column to the Scoring Thresholds table. It allows for the setting of individual scoring thresholds for modified peptides. For RTS, this will be populated from the list of variable/static modifications in the database search settings.

Default: Deselected

NOTE: In the Scoring Threshold table, you can select multiple modifications per row by pressing CTRL + Left mouse click.

Enable Close-Out

Default: Deselected

Maximum Closeout Triggers

(Available when Enable Close-Out is selected.)

Maximum number of dependent scan triggers for a given library spectrum or its protein group. After this number is reached, no more dependent scans will be triggered for that item.

Default: 10

Enable Protein Close-Out

(Available when Enable Close-Out is selected.)

When enabled, the system will stop triggering additional scan events for a protein once the defined threshold is met.

Signal to Noise Threshold

The summed TMT reporter signal to noise threshold required for an MS³ scan to increment the identified library spectrums's close-out counter.

Range: 0–1E+20: default: 100

Add Adducts to Dynamic Exclusion

(Available in Small Molecule application mode.)

When filter thresholds pass, add all considered adduct masses to dynamic exclusion.

When enabled and the search produced a passing result, the filter will add all considered adduct query m/z values to dynamic exclusion.

Default: Deselected

Add Matched Fragments to Dynamic Exclusion

When enabled, matched fragments are added to dynamic exclusion.

When enabled, each matched fragment from the experimental spectrum found in the top library result is added to dynamic exclusion.

Default: Deselected

Add Passing Search Results to Dynamic Exclusion

When enabled, passing search results will be added to a search dynamic exclusion list.

When operating under a targeted scan (tMS²) with Multi-Precursor Search enabled, items will be excluded based on their sequence (or compound name) and charge state. When operating in a DDA mode using Multi-Precursor Search, passing search results will be added to the dynamic exclusion filter preceding the MS² scan.

Default: Deselected

The following parameters are available when this option is checked:

• Exclusion duration (s): The amount of time the precursor remains on the exclusion list.
• Range: 0–6,000; default: 60
• Mass Tolerance: The mass tolerance can be expressed as parts per million (ppm) or m/z.
• Low: The low side of the mass tolerance width window.
• Range (ppm): 0.01–1,000,000; default: 10
  Range (m/z): 0.01–100; default: 0.5
• High: The high side of the mass tolerance width window.
• Range (ppm): 0.01–1,000,000; default: 10
  Range (m/z): 0.01–100; default: 0.5

Scoring Thresholds table

Add, delete, import, or export threshold values.

Scoring thresholds are applied on each search result. The spectrum passes through the filter if the scores for a spectrum are higher than the indicated thresholds.

Compound Class Filter

Add, delete, import, or export compound class filter values.

Promotes—or rejects—mzVault annotated compound classes to override the threshold parameter settings. Compound classes that are set to “Promote” trigger downstream scans; compound classes that are set to “Reject” do not.