SDRF-Proteomics: Crosslinking Mass Spectrometry Template

1. Status of this Template

This document provides a template for annotating crosslinking mass spectrometry (XL-MS) experiments in SDRF-Proteomics format. This template defines additional columns that can be combined with any sample template (human, vertebrates, etc.) to capture crosslinking-specific metadata.

Type: Experiment Template (used in combination with sample templates)

Status: Stable

Version: 1.0.0 - 2026-01

2. Abstract

Crosslinking mass spectrometry (XL-MS) uses chemical reagents to covalently link amino acid residues in close spatial proximity, providing information for protein structure determination, protein-protein interaction mapping, complex characterization, and integrative structural biology.

This template is designed with data analysis in mind. The REQUIRED columns capture the minimum metadata needed to configure XL-MS search engines like Scout, xiSEARCH, pLink2, or MeroX. Sample preparation details (reaction time, temperature, concentration) are captured as OPTIONAL columns for reproducibility documentation.

3. Column Checklist

3.1. REQUIRED Columns

These columns are essential for configuring XL-MS analysis workflows:

Column Name	Type	Description	Example Values
characteristics[enrichment process]	Sample	Indicates the sample was enriched for crosslinked peptides (PRIDE:0000838). This is a sample-level property.	enrichment of cross-linked peptides
comment[cross-linker]	Data acquisition	Cross-linker compound with structured properties for analysis tools. Uses XLMOD ontology (parent term XLMOD:00004). Format: NT=name;AC=accession;CL=cleavable;TA=targets;MH/ML=stub masses	NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01;ML=85.98
comment[dissociation method]	Data acquisition	Fragmentation method used in MS2. Critical for cleavable crosslinkers (DSSO, DSBU) which generate diagnostic stub ions.	HCD, CID, ETD, EThcD, stepped HCD

Column Name

Type

Description

Example Values

characteristics[enrichment process]

Sample

Indicates the sample was enriched for crosslinked peptides (PRIDE:0000838). This is a sample-level property.

enrichment of cross-linked peptides

comment[cross-linker]

Data acquisition

Cross-linker compound with structured properties for analysis tools. Uses XLMOD ontology (parent term XLMOD:00004). Format: NT=name;AC=accession;CL=cleavable;TA=targets;MH/ML=stub masses

NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01;ML=85.98

comment[dissociation method]

Data acquisition

Fragmentation method used in MS2. Critical for cleavable crosslinkers (DSSO, DSBU) which generate diagnostic stub ions.

HCD, CID, ETD, EThcD, stepped HCD

Note	Columns inherited from ms-proteomics are also required: `comment[cleavage agent details]`, `comment[instrument]`, `comment[label]`, `comment[fraction identifier]`.

3.2. Column Type Distinction

characteristics[…] = Sample properties (what IS the sample)
comment[…] = Data acquisition properties (how was data acquired)

For crosslinking:

characteristics[enrichment process] = the sample underwent enrichment for crosslinked peptides (PRIDE:0000838, sample property)
comment[cross-linker] = which crosslinker was used and its properties (data acquisition)

3.3. Template Selection: When to Apply Human Template

The human template should only be applied to biological human samples (tissues, cell lines, body fluids). It should NOT be applied to:

Recombinant protein standards with human sequences
Synthetic protein mixtures for benchmarking
In-vitro crosslinked purified proteins

For these non-biological samples:

Sample Type	Templates	characteristics[organism]	characteristics[material type]
Human tissue/cells	ms-proteomics, crosslinking, human	homo sapiens	tissue, cell line
Recombinant protein standard	ms-proteomics, crosslinking	homo sapiens	recombinant protein
Synthetic benchmark	ms-proteomics, crosslinking	homo sapiens (or source species)	synthetic construct

Sample Type

Templates

characteristics[organism]

characteristics[material type]

Human tissue/cells

ms-proteomics, crosslinking, human

homo sapiens

tissue, cell line

Recombinant protein standard

ms-proteomics, crosslinking

homo sapiens

recombinant protein

Synthetic benchmark

ms-proteomics, crosslinking

homo sapiens (or source species)

synthetic construct

Note	The `characteristics[organism]` indicates the proteome to search against, not necessarily the biological origin of the sample. Use `characteristics[material type]` to clarify the sample nature.

3.4. File Metadata Columns

SDRF files SHOULD include metadata columns to specify version and template information. Only list leaf templates; parent templates are implied by inheritance (crosslinking inherits from ms-proteomics). Place these columns at the end of the file.

Non-biological samples (recombinant proteins, synthetic constructs):

…	comment[sdrf version]	comment[sdrf template]
…	v1.1.0	crosslinking v1.0.0

…

comment[sdrf version]

comment[sdrf template]

…

v1.1.0

crosslinking v1.0.0

Human biological samples (tissues, cell lines) - use multiple template columns:

…	comment[sdrf version]	comment[sdrf template]	comment[sdrf template]
…	v1.1.0	human v1.1.0	crosslinking v1.0.0

…

comment[sdrf version]

comment[sdrf template]

…

v1.1.0

human v1.1.0

crosslinking v1.0.0

Each template’s version indicates which version of that specific template the file conforms to.

3.5. RECOMMENDED Columns

Column Name	Description	Example Values
comment[collision energy]	Collision energy used for fragmentation. Important for cleavable crosslinker analysis.	27, 30%, stepped 27+-6%
comment[crosslink enrichment method]	Method used to enrich crosslinked peptides before MS analysis.	size exclusion chromatography, strong cation exchange chromatography, FAIMS
comment[modification parameters]	Post-translational modifications (inherited from base template).	NT=Carbamidomethyl;AC=UNIMOD:4;TA=C;MT=Fixed

Column Name

Description

Example Values

comment[collision energy]

Collision energy used for fragmentation. Important for cleavable crosslinker analysis.

27, 30%, stepped 27+-6%

comment[crosslink enrichment method]

Method used to enrich crosslinked peptides before MS analysis.

size exclusion chromatography, strong cation exchange chromatography, FAIMS

comment[modification parameters]

Post-translational modifications (inherited from base template).

NT=Carbamidomethyl;AC=UNIMOD:4;TA=C;MT=Fixed

3.6. OPTIONAL Columns (Sample Preparation Details)

These columns document sample preparation for reproducibility but are not required for analysis:

Column Name	Description	Example Values
characteristics[crosslink type]	Type of crosslinker chemistry (derivable from reagent).	homo-bifunctional, hetero-bifunctional, zero-length, MS-cleavable
characteristics[crosslink distance]	Maximum Ca-Ca distance constraint (for structural interpretation).	30 A, 26.4 A, 11.4 A
characteristics[enrichment process]	Whether sample was enriched for crosslinked peptides.	enrichment of cross-linked peptides, no enrichment
comment[crosslinker concentration]	Concentration of crosslinking reagent used.	2 mM, 500 uM, 1 mM
characteristics[crosslinking reaction time]	Duration of the crosslinking reaction.	30 min, 1 h, 45 min
characteristics[crosslinking temperature]	Temperature at which crosslinking was performed.	25 C, 4 C, room temperature
comment[crosslinker to protein ratio]	Molar ratio of crosslinker to protein.	50:1, 100:1
comment[quenching reagent]	Reagent used to quench the crosslinking reaction.	Tris-HCl, ammonium bicarbonate, glycine

Column Name

Description

Example Values

characteristics[crosslink type]

Type of crosslinker chemistry (derivable from reagent).

homo-bifunctional, hetero-bifunctional, zero-length, MS-cleavable

characteristics[crosslink distance]

Maximum Ca-Ca distance constraint (for structural interpretation).

30 A, 26.4 A, 11.4 A

characteristics[enrichment process]

Whether sample was enriched for crosslinked peptides.

enrichment of cross-linked peptides, no enrichment

comment[crosslinker concentration]

Concentration of crosslinking reagent used.

2 mM, 500 uM, 1 mM

characteristics[crosslinking reaction time]

Duration of the crosslinking reaction.

30 min, 1 h, 45 min

characteristics[crosslinking temperature]

Temperature at which crosslinking was performed.

25 C, 4 C, room temperature

comment[crosslinker to protein ratio]

Molar ratio of crosslinker to protein.

50:1, 100:1

comment[quenching reagent]

Reagent used to quench the crosslinking reaction.

Tris-HCl, ammonium bicarbonate, glycine

4. Crosslinker Annotation Format

The comment[cross-linker] column uses a structured key-value format that enables automated analysis configuration. The accession should reference terms from the XLMOD ontology (parent term XLMOD:00004 - cross-linker):

NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01;ML=85.98

4.1. Key-Value Properties

Key	Property	Status	Description	Example
NT	Name	REQUIRED	Cross-linker name	NT=DSSO
AC	Accession	REQUIRED	XLMOD or ChEBI accession	AC=XLMOD:02010
CL	Cleavable	REQUIRED	MS-cleavable (yes/no)	CL=yes
TA	Target Amino Acids	RECOMMENDED	Residues targeted by crosslinker	TA=K,S,T,Y,nterm
MH	Heavy Stub Mass	REQUIRED (if cleavable)	Mass of heavy fragment (Da)	MH=54.01
ML	Light Stub Mass	REQUIRED (if cleavable)	Mass of light fragment (Da)	ML=85.98
SM	Spacer Mass	REQUIRED (if non-cleavable, except zero-length)	Full crosslinker mass (Da). For zero-length crosslinkers (e.g., EDC), SM=0 and can be omitted.	SM=138.07

Key

Property

Status

Description

Example

Name

REQUIRED

Cross-linker name

NT=DSSO

Accession

REQUIRED

XLMOD or ChEBI accession

AC=XLMOD:02010

Cleavable

REQUIRED

MS-cleavable (yes/no)

CL=yes

Target Amino Acids

RECOMMENDED

Residues targeted by crosslinker

TA=K,S,T,Y,nterm

Heavy Stub Mass

REQUIRED (if cleavable)

Mass of heavy fragment (Da)

MH=54.01

Light Stub Mass

REQUIRED (if cleavable)

Mass of light fragment (Da)

ML=85.98

Spacer Mass

REQUIRED (if non-cleavable, except zero-length)

Full crosslinker mass (Da). For zero-length crosslinkers (e.g., EDC), SM=0 and can be omitted.

SM=138.07

4.2. Common Crosslinker Examples

# MS-cleavable crosslinkers (Scout defaults)
NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01056468;ML=85.98263585
NT=DSBU;AC=XLMOD:02011;CL=yes;TA=K,S,T,Y,nterm;MH=85.05276381;ML=111.03202835
NT=DSBSO;AC=XLMOD:02012;CL=yes;TA=K,S,T,Y,nterm;MH=54.01056;ML=236.0177
NT=DSAU;AC=XLMOD:02013;CL=yes;TA=K,S,T,Y,nterm;MH=57.02146371;ML=83.00072827

# Non-cleavable crosslinkers
NT=DSS;AC=XLMOD:02001;CL=no;TA=K,S,T,Y,nterm;SM=138.07
NT=BS3;AC=XLMOD:02000;CL=no;TA=K,S,T,Y,nterm;SM=138.07

# Zero-length crosslinker (SM=0 is implicit for zero-length)
NT=EDC;AC=XLMOD:02009;CL=no;TA=K,D,E;SM=0

# Photo-reactive crosslinker
NT=SDA;AC=XLMOD:02005;CL=no;TA=K,S,T,Y,nterm

4.3. Crosslinker Reference Table

Crosslinker	Type	Target Residues	Cleavable	Spacer Arm	Max Ca-Ca
DSSO	Homo-bifunctional, NHS ester	K, S, T, Y, N-term	Yes	10.3 A	~26 A
DSBU	Homo-bifunctional, NHS ester	K, S, T, Y, N-term	Yes	12.5 A	~30 A
DSBSO	Homo-bifunctional, NHS ester	K, S, T, Y, N-term	Yes	10.3 A	~25 A
DSAU	Homo-bifunctional, NHS ester	K, S, T, Y, N-term	Yes	10.0 A	~25 A
DSS	Homo-bifunctional, NHS ester	K, S, T, Y, N-term	No	11.4 A	~30 A
BS3	Homo-bifunctional, NHS ester (water-soluble)	K, S, T, Y, N-term	No	11.4 A	~30 A
EDC	Zero-length	D, E to K	No	0 A	~15 A
SDA	Photo-reactive	K + any	No	3.9 A	~20 A

Crosslinker

Type

Target Residues

Cleavable

Spacer Arm

Max Ca-Ca

DSSO

Homo-bifunctional, NHS ester

K, S, T, Y, N-term

Yes

10.3 A

~26 A

DSBU

Homo-bifunctional, NHS ester

K, S, T, Y, N-term

Yes

12.5 A

~30 A

DSBSO

Homo-bifunctional, NHS ester

K, S, T, Y, N-term

Yes

10.3 A

~25 A

DSAU

Homo-bifunctional, NHS ester

K, S, T, Y, N-term

Yes

10.0 A

~25 A

DSS

Homo-bifunctional, NHS ester

K, S, T, Y, N-term

11.4 A

~30 A

BS3

Homo-bifunctional, NHS ester (water-soluble)

K, S, T, Y, N-term

11.4 A

~30 A

EDC

Zero-length

D, E to K

0 A

~15 A

SDA

Photo-reactive

K + any

3.9 A

~20 A

5. Example Project

A complete annotated crosslinking dataset is available:

PXD042173 - Proteome-scale recombinant standard datasets for XL-MS

Crosslinker: DSSO (MS-cleavable)
Instrument: Orbitrap Fusion Lumos with FAIMS
Enrichment: Strong cation exchange chromatography
Fragmentation: Stepped HCD (27+-6%)
Material Type: Recombinant protein standard (not a biological sample)

Open in SDRF Viewer | Download SDRF file

6. Example SDRF Structure

source name	characteristics[organism]	characteristics[material type]	characteristics[enrichment process]	comment[cross-linker]	comment[dissociation method]	comment[collision energy]	comment[data file]
batch1_sample1	homo sapiens	recombinant protein	enrichment of cross-linked peptides	NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01;ML=85.98	HCD	stepped 27+-6%	sample1_fraction1.raw
batch1_sample2	homo sapiens	recombinant protein	enrichment of cross-linked peptides	NT=DSSO;AC=XLMOD:02010;CL=yes;TA=K,S,T,Y,nterm;MH=54.01;ML=85.98	HCD	stepped 27+-6%	sample1_fraction2.raw

Sample metadata Data acquisition metadata

7. Mapping to Analysis Tools

This section shows how SDRF columns map to parameters in common XL-MS search engines:

7.1. Scout

SDRF Column	Scout Parameter	Notes
comment[cross-linker] (NT)	CXLReagent.Name	Crosslinker name
comment[cross-linker] (TA)	CXLReagent.Targets	Target residues (K, S, T, Y)
comment[cross-linker] (MH)	CXLReagent.HeavyFragment	Heavy stub mass for cleavable XL
comment[cross-linker] (ML)	CXLReagent.LightFragment	Light stub mass for cleavable XL
comment[cleavage agent details]	Enzyme	Trypsin, Lys-C, etc.
comment[modification parameters]	StaticModifications, VariableModifications	Carbamidomethyl, Oxidation, etc.

SDRF Column

Scout Parameter

Notes

comment[cross-linker] (NT)

CXLReagent.Name

Crosslinker name

comment[cross-linker] (TA)

CXLReagent.Targets

Target residues (K, S, T, Y)

comment[cross-linker] (MH)

CXLReagent.HeavyFragment

Heavy stub mass for cleavable XL

comment[cross-linker] (ML)

CXLReagent.LightFragment

Light stub mass for cleavable XL

comment[cleavage agent details]

Enzyme

Trypsin, Lys-C, etc.

comment[modification parameters]

StaticModifications, VariableModifications

Carbamidomethyl, Oxidation, etc.

7.2. xiSEARCH

SDRF Column	xiSEARCH Parameter	Notes
comment[cross-linker]	crosslinker: definition	Full crosslinker config
comment[dissociation method]	Fragment ions considered	HCD enables stub ion detection
comment[cleavage agent details]	digestion: config	Enzyme definition
comment[modification parameters]	modification: config	Fixed and variable mods

SDRF Column

xiSEARCH Parameter

Notes

comment[cross-linker]

crosslinker: definition

Full crosslinker config

comment[dissociation method]

Fragment ions considered

HCD enables stub ion detection

comment[cleavage agent details]

digestion: config

Enzyme definition

comment[modification parameters]

modification: config

Fixed and variable mods

8. Validation

Crosslinking SDRF files should be validated using sdrf-pipelines:

pip install sdrf-pipelines

# Validate with crosslinking template
parse_sdrf validate-sdrf --sdrf_file your_file.sdrf.tsv \
  --template ms-proteomics \
  --template crosslinking

# For human samples
parse_sdrf validate-sdrf --sdrf_file your_file.sdrf.tsv \
  --template ms-proteomics \
  --template human \
  --template crosslinking

9. References

Leitner A, et al. (2016) Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines. Trends in Biochemical Sciences.
O’Reilly FJ, Rappsilber J. (2018) Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology. Nature Structural & Molecular Biology.
Iacobucci C, et al. (2019) A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software. Nature Protocols.
XLMOD database: https://www.ebi.ac.uk/ols4/ontologies/xlmod
Scout: https://github.com/diogobor/Scout
xiSEARCH: https://www.rappsilberlab.org/software/xisearch/