Table 8.
Settings for the protein-level and malonyl-level analysis in Spectronaut
| Protein-level analysis | Malonyl-level analysis | ||
|---|---|---|---|
| Pulsar search | |||
| Peptides | Enzymes/Cleavage Rules | Trypsin/P | Trypsin/P |
| Digest Type | Specific | Specific | |
| Missed Cleavagesa | 2 | 4 | |
| Modifications | Max Variable Modificationsa | 5 | 8 |
| Fixed Modifications | Carbamidomethyl (C) | Carbamidomethyl (C) | |
| Variable Modificationsa | Acetyl (Protein N-term) Oxidation (M) |
Acetyl (Protein N-term) Oxidation (M) Malonyl (K) |
|
| Identification | PSM FDR | 0.01 | 0.01 |
| Peptide FDR | 0.01 | 0.01 | |
| Protein Group FDR | 0.01 | 0.01 | |
| Result Filters |
Fragment Ions | ||
| Ion AA Length (min) | N = 3 | N = 3 | |
| m/z | 200 - 3,000 | 200 - 3,000 | |
| Relative Intensity | Min = 5 | Min = 5 | |
| Precursors | |||
| Best N Fragments per Peptide | 3–6 | 3–6 | |
| Modifications | None | None | |
| DIA analysis | |||
| XIC Extraction | MS1 Mass Tolerance Strategy | Dynamic | Dynamic |
| Correction Factor | 1 | 1 | |
| MS2 Mass Tolerance Strategy | Dynamic | Dynamic | |
| Correction Factor | 1 | 1 | |
| Calibration | MZ Extraction Strategy | Maximum Intensity | Maximum Intensity |
| Allow source-specific iRT Calibration | True | True | |
| Precision iRT | True | True | |
| iRT <-> RT Regression Type | Local (Non-Linear) Regression | Local (Non-Linear) Regression | |
| MS1 Mass Tolerance Strategy | System Default | System Default | |
| MS2 Mass Tolerance Strategy | System Default | System Default | |
| Identification | Precursor PEP Cutoffa | 0.2 | 1 |
| Precursor Qvalue Cutoff | 0.01 | 0.01 | |
| Protein Qvalue Cutoff (Experiment) | 0.01 | 0.01 | |
| Protein Qvalue Cutoff (Run)a | 0.05 | 1 | |
| Exclude Duplicate Assays | True | True | |
| Generate Decoys | True | True | |
| Decoy Generation Method | Mutated | Mutated | |
| Preferred Fragment Source | NN Predicted Fragments | NN Predicted Fragments | |
| Decoy Limit Strategy | Dynamic | Dynamic | |
| Library Size Fraction | 0.1 | 0.1 | |
| Quantification | Precursor Strategy | Identified (QValue) | Identified (QValue) |
| Imputation Strategy | Use Background Signal | Use Background Signal | |
| Quantify MS Level | MS2 | MS2 | |
| Quantify Type | Area | Area | |
| Cross-Run Normalizationa | True | None | |
| Normalization Filter Typea | None | N/A | |
| Normalization Strategya | Local Normalization | N/A | |
| Row Selectiona | Qvalue sparse | N/A | |
| Interference Correction | True | True | |
| Only Identified Peptides | True | True | |
| MS1 Min | 2 | 2 | |
| MS2 Min | 3 | 3 | |
| Major (Protein) Groupinga | by Protein Group Id | by Protein Group Id | |
| Major Group Quantitya | Sum peptide quantity | Mean peptide quantity | |
| Major Group Top Na | Min = 1; Max = 7 | Min = 1; Max = 3 | |
| Minor (Peptide) Groupinga | by Stripped Sequence | by Modified Sequence | |
| Minor Group Quantitya | Sum precursor quantity | Mean precursor quantity | |
| Minor Group Top Na | Min = 1; Max = 10 | Min = 1; Max = 3 | |
| Workflow | Profiling Strategya | iRT Profiling | None |
| Carry-over exact Peak Boundariesa | None | N/A | |
| Profiling Row Selectiona | Minimum QValue Row Selection | N/A | |
| Qvalue Thresholda | 0.01 | N/A | |
| Profiling Target Selectiona | Automatic Selection | N/A | |
| PTM Workflow | PTM Localizationa | None | True |
| Probability Cutoffa | N/A | 0.75 | |
| PTM Analysisa | N/A | True | |
| Multiplicitya | N/A | True | |
| Flanking Regiona | N/A | 7 | |
| PTM Consolidationa | N/A | Sum | |
a
Parameters that differ between the protein-level and malonyl-level analysis.