fix: improve accuracy of preliminary score estimation

- Remove fragment_min/max_mz parameters
- Adjust ppm tolerance during preliminary search to account for charge

Author: lazear

Diff for: CHANGELOG.md

@@ -4,6 +4,13 @@ All notable changes to this project will be documented in this file.
 The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/),
 and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html).
 
+## [v0.15.0-alpha] (unreleased)
+### Added
+- Initial support for searching diaPASEF data
+### Changed
+- Don't deisotope reporter ion regions if MS2-based TMT/iTRAQ is used
+- Removed `fragment_min_mz` and `fragment_max_mz` parameters. These were decreasing the accuracy of preliminary scoring estimation when attempting to annotate multiply-charged, high-m/z ions.
+
 ## [v0.14.7]
 ### Added
 - Added columns missing from parquet output: `semi_enzymatic` and `missed_cleavages`

Diff for: Cargo.lock

@@ -184,21 +184,16 @@ impl Runner {
             .sn
             .then_some(self.parameters.quant.tmt_settings.level);
 
-        let (min_fragment_mz, min_deisotope_mz) = match &self.parameters.quant.tmt {
+        let min_deisotope_mz = match &self.parameters.quant.tmt {
             Some(i) => match self.parameters.quant.tmt_settings.level {
-                2 => (
-                    i.reporter_masses().first().map(|x| x * (1.0 - 20E-6)),
-                    i.reporter_masses().last().map(|x| x * (1.0 + 20E-6)),
-                ),
-                _ => (None, None),
+                2 => i.reporter_masses().last().map(|x| x * (1.0 + 20E-6)),
+                _ => None,
             },
-            None => (None, None),
+            None => None,
         };
 
         let sp = SpectrumProcessor::new(
             self.parameters.max_peaks,
-            min_fragment_mz.unwrap_or(self.parameters.database.fragment_min_mz),
-            self.parameters.database.fragment_max_mz,
             self.parameters.deisotope,
             min_deisotope_mz.unwrap_or(0.0),
         );
@@ -268,8 +263,6 @@ impl Runner {
             min_precursor_charge: self.parameters.precursor_charge.0,
             max_precursor_charge: self.parameters.precursor_charge.1,
             max_fragment_charge: self.parameters.max_fragment_charge,
-            min_fragment_mass: self.parameters.database.fragment_min_mz,
-            max_fragment_mass: self.parameters.database.fragment_max_mz,
             chimera: self.parameters.chimera,
             report_psms: self.parameters.report_psms,
             wide_window: self.parameters.wide_window,

Diff for: crates/sage-cli/src/main.rs

@@ -13,7 +13,7 @@ fn integration() -> anyhow::Result<()> {
     let spectra = sage_cloudpath::util::read_mzml("../../tests/LQSRPAAPPAPGPGQLTLR.mzML", 0, None)?;
     assert_eq!(spectra.len(), 1);
 
-    let sp = SpectrumProcessor::new(100, 0.0, 1500.0, true, 0.0);
+    let sp = SpectrumProcessor::new(100, true, 0.0);
     let processed = sp.process(spectra[0].clone());
     assert!(processed.peaks.len() <= 300);
 
@@ -27,8 +27,6 @@ fn integration() -> anyhow::Result<()> {
         min_precursor_charge: 2,
         max_precursor_charge: 4,
         max_fragment_charge: Some(1),
-        min_fragment_mass: 0.0,
-        max_fragment_mass: 1500.0,
         chimera: false,
         report_psms: 1,
         wide_window: false,

Diff for: crates/sage-cli/tests/integration.rs

@@ -1,6 +1,6 @@
 [package]
 name = "sage-cloudpath"
-version = "0.14.7"
+version = "0.15.0-alpha"
 authors = ["Michael Lazear <[email protected]"]
 edition = "2021"
 rust-version = "1.62"

Diff for: crates/sage-cloudpath/Cargo.toml

@@ -1,6 +1,6 @@
 [package]
 name = "sage-core"
-version = "0.14.7"
+version = "0.15.0-alpha"
 authors = ["Michael Lazear <[email protected]"]
 edition = "2021"
 rust-version = "1.62"

Diff for: crates/sage/Cargo.toml

@@ -63,10 +63,6 @@ pub struct Builder {
     pub bucket_size: Option<usize>,
 
     pub enzyme: Option<EnzymeBuilder>,
-    /// Minimum fragment m/z that will be stored in the database
-    pub fragment_min_mz: Option<f32>,
-    /// Maximum fragment m/z that will be stored in the database
-    pub fragment_max_mz: Option<f32>,
     /// Minimum peptide monoisotopic mass that will be fragmented
     pub peptide_min_mass: Option<f32>,
     /// Maximum peptide monoisotopic mass that will be fragmented
@@ -95,8 +91,6 @@ impl Builder {
         let bucket_size = self.bucket_size.unwrap_or(8192).next_power_of_two();
         Parameters {
             bucket_size,
-            fragment_min_mz: self.fragment_min_mz.unwrap_or(150.0),
-            fragment_max_mz: self.fragment_max_mz.unwrap_or(2000.0),
             peptide_min_mass: self.peptide_min_mass.unwrap_or(500.0),
             peptide_max_mass: self.peptide_max_mass.unwrap_or(5000.0),
             ion_kinds: self.ion_kinds.unwrap_or(vec![Kind::B, Kind::Y]),
@@ -120,8 +114,6 @@ impl Builder {
 pub struct Parameters {
     pub bucket_size: usize,
     pub enzyme: EnzymeBuilder,
-    pub fragment_min_mz: f32,
-    pub fragment_max_mz: f32,
     pub peptide_min_mass: f32,
     pub peptide_max_mass: f32,
     pub ion_kinds: Vec<Kind>,
@@ -236,8 +228,6 @@ impl Parameters {
                             }
                         };
                         ion_idx_filter
-                            && ion.monoisotopic_mass >= self.fragment_min_mz
-                            && ion.monoisotopic_mass <= self.fragment_max_mz
                     })
                     .map(move |(_, ion)| Theoretical {
                         peptide_index: PeptideIx(idx as u32),
@@ -426,8 +416,17 @@ pub struct IndexedQuery<'d> {
 
 impl<'d> IndexedQuery<'d> {
     /// Search for a specified `fragment_mz` within the database
-    pub fn page_search(&self, fragment_mz: f32) -> impl Iterator<Item = &Theoretical> {
-        let (fragment_lo, fragment_hi) = self.fragment_tol.bounds(fragment_mz);
+    pub fn page_search(&self, fragment_mz: f32, charge: u8) -> impl Iterator<Item = &Theoretical> {
+        let mass = fragment_mz * charge as f32;
+
+        // Account for multiplication of observed decharged mass
+        // - relative tolerance needs to be proportionally decreased
+        let tol = match self.fragment_tol {
+            Tolerance::Ppm(lo, hi) => Tolerance::Ppm(lo / charge as f32, hi / charge as f32),
+            Tolerance::Da(_, _) => self.fragment_tol,
+        };
+
+        let (fragment_lo, fragment_hi) = tol.bounds(mass);
         let (precursor_lo, precursor_hi) = self.precursor_tol.bounds(self.precursor_mass);
 
         // Locate the left and right page indices that contain matching fragments
@@ -587,8 +586,6 @@ mod test {
                 max_len: Some(10),
                 ..Default::default()
             },
-            fragment_min_mz: 100.0,
-            fragment_max_mz: 1000.0,
             peptide_min_mass: 150.0,
             peptide_max_mass: 5000.0,
             ion_kinds: vec![Kind::B, Kind::Y],

Diff for: crates/sage/src/database.rs

@@ -183,8 +183,6 @@ pub struct Scorer<'db> {
     pub min_precursor_charge: u8,
     pub max_precursor_charge: u8,
     pub max_fragment_charge: Option<u8>,
-    pub min_fragment_mass: f32,
-    pub max_fragment_mass: f32,
     pub chimera: bool,
     pub report_psms: usize,
 
@@ -270,8 +268,7 @@ impl<'db> Scorer<'db> {
 
         for peak in query.peaks.iter() {
             for charge in 1..max_fragment_charge {
-                let mass = peak.mass * charge as f32;
-                for frag in candidates.page_search(mass) {
+                for frag in candidates.page_search(peak.mass, charge) {
                     let idx = frag.peptide_index.0 as usize - candidates.pre_idx_lo;
                     let sc = &mut hits.preliminary[idx];
                     if sc.matched == 0 {
@@ -280,6 +277,7 @@ impl<'db> Scorer<'db> {
                         sc.peptide = frag.peptide_index;
                         sc.isotope_error = isotope_error;
                     }
+
                     sc.matched += 1;
                     hits.matched_peaks += 1;
                 }
@@ -598,6 +596,7 @@ impl<'db> Scorer<'db> {
             for charge in 1..max_fragment_charge {
                 // Experimental peaks are multipled by charge, therefore theoretical are divided
                 let mz = frag.monoisotopic_mass / charge as f32;
+
                 if let Some(peak) = crate::spectrum::select_most_intense_peak(
                     &query.peaks,
                     mz,

Diff for: crates/sage/src/scoring.rs

@@ -39,8 +39,6 @@ pub struct Deisotoped {
 #[derive(Debug, Clone)]
 pub struct SpectrumProcessor {
     pub take_top_n: usize,
-    pub max_fragment_mz: f32,
-    pub min_fragment_mz: f32,
     pub min_deisotope_mz: f32,
     pub deisotope: bool,
 }
@@ -262,17 +260,9 @@ impl SpectrumProcessor {
     /// * `min_fragment_mz`: Keep only fragments >= this m/z
     /// * `max_fragment_mz`: Keep only fragments <= this m/z
     /// * `deisotope`: Perform deisotoping & charge state deconvolution
-    pub fn new(
-        take_top_n: usize,
-        min_fragment_mz: f32,
-        max_fragment_mz: f32,
-        deisotope: bool,
-        min_deisotope_mz: f32,
-    ) -> Self {
+    pub fn new(take_top_n: usize, deisotope: bool, min_deisotope_mz: f32) -> Self {
         Self {
             take_top_n,
-            min_fragment_mz,
-            max_fragment_mz,
             min_deisotope_mz,
             deisotope,
         }
@@ -310,11 +300,7 @@ impl SpectrumProcessor {
 
             peaks
                 .into_iter()
-                .filter(|peak| {
-                    peak.envelope.is_none()
-                        && peak.mz >= self.min_fragment_mz
-                        && peak.mz <= self.max_fragment_mz
-                })
+                .filter(|peak| peak.envelope.is_none())
                 .map(|peak| {
                     // Convert from MH* to M
                     let mass = (peak.mz - PROTON) * peak.charge.unwrap_or(1) as f32;
@@ -330,7 +316,6 @@ impl SpectrumProcessor {
                 .mz
                 .iter()
                 .zip(spectrum.intensity.iter())
-                .filter(|&(mz, _)| *mz >= self.min_fragment_mz && *mz <= self.max_fragment_mz)
                 .map(|(mz, &intensity)| {
                     let mass = (mz - PROTON) * 1.0;
                     Peak { mass, intensity }

Diff for: crates/sage/src/spectrum.rs

@@ -61,7 +61,7 @@ fn check_all_ions_visited(target_fragment_mz: f32, bucket_size: usize) {
     // are returned to us by searching the database.
     let query = database.query(1000.0, Tolerance::Da(-5000.0, 5000.0), fragment_tol);
 
-    for fragment in query.page_search(target_fragment_mz) {
+    for fragment in query.page_search(target_fragment_mz, 1) {
         visited[fragment.peptide_index.0 as usize] += 1;
     }