Cross-linking Proteomics Analysis Tutorial

• Requirements
• Preparation
• Target Selection
• Target Binding
• Analysis Reports
• Interactive Visualization

Requirements

• traditional mass spectrometry data, e.g., DDA.raw
• PSM search results of pLink, e.g., DDA.psm.csv
• filtered PSM search results of pLink, e.g., ****.filteredcross-linkedspectra.csv

Preparation

MS Data Format

The raw data should be converted into an open-source format such as MS1/MS2. Using ThermoRawRead is recommended.

PSM

The PSM list can be used as-is. If you are interested in specific identifications, e.g., phosphopeptide, it’s ok to filter the list manually.

Target Selection

TargetWizard provides the TargetSelect feature to generate a target list (a.k.a., inclusion list) for further MS data acquisition. As shown in the following GUI, you can filter the PSM list by FDR value, and/or Target-Decoy type.

The parameters are described as below:

Data

MS Data

PSM

unfiltered PSM level identification results.

Task Name

appeared in output results.

Max. MS1 Mass Error

used to match PSMs and MS1 peaks.

FDR Range

used to filter PSMs. Inf and -Inf are also valid. For example, -Inf % ≤ FDR ≤ Inf % will not remove any PSM.

Target / Decoy Type

TT for Target-Target, TD for Target-Decoy, DD for Decoy-Decoy.

Inter- / Intra-Protein

Protein type of crosslinks: inter-protein or intra-protein.

Batch Size

Max batch size of each data acquisition size. Inf is also valid. It controls the load of each data acquisition, and TargetWizard will split the task into batches automatically.

RT Window

Retention time window size for targeted acquisition. The RT values are estimated using PSM and MS1. The size is the total length, instead of radius.

LC Length

LC Length of the LC-MS/MS experiment. Inf is also valid. RT values will be trimmed using the length.

List Format

TargetWizard Support three formats:

• TargetWizard: most detailed format, including many useful information.
• Thermo Q Exactive: format can be used directly by Thermo Q Exactive instrument.
• Thermo Fusion: format can be used directly by Thermo Fusion instrument.

Output Directory

where output results are saved to.

Target Binding

TargetWizard provides three strategies to bind targets and MS data acquired using the target list. The feature exports various MS formats including MS2, MGF, and PF2, which can be used for further analysis. You can use the exported data for further identification. The matched targets are used as precursor ions of these MS2 scans.

The parameters are described as below:

Targeted MS Data

MS data acquired using the target list

Target List

previously generated list of interested targets.

List Format

Four options are available:

• Auto Detect
• TargetWizard
• Thermo Q Exactive
• Thermo Fusion

Binding Mode

Three options are available:

• by isolation center: match targets and MS2 scans when targets are isolation centers of these MS2 scans.
• by isolation window: match targets and MS2 scans when targets are in isolation windows of these MS2 scans.
• by extended isolation window: similar to by isolation window but extend the windows to the left by 1 Th so that incompleted precursor isotope clusters can also be considered for identification.

Max. RTime Error

When setting the error to zero, only match targets and MS2 scans when scans are in the RT window of the targets. The RT window will be extended by setting the error to larger values.

Max. Mass Error

Mass Error for targets and MS2 scans. Used when the mode is set to by isolation center only.

Output Format

• CSV
• TSV
• MS2
• MGF
• PF2

Output Directory

where output results are saved to.

Analysis Reports

TargetWizard can generate various reports automatically to assist with targeted proteomics analysis.

• Basic Acquisition Report
• Target Selection Report
• Target Acquisition Cross-linked Peptide Report
• Cross-linked Peptide Coverage Report
• Comparative Cross-link Signal-to-Noise Ratio Report

For details of these reports, please see the manual.

Interactive Visualization

In addition to the static reports, TargetWizard provides an interactive visualization feature to help with insightful case studies.

• Regular Cross-linked Peptide View
• Comparative Cross-linked Peptide View
• Cross-link Site View
• Cross-link Exhaustive Search View

For details of these reports, please see the manual.