In vivo Cross-Linking Mass Spectrometry for Protein and Complex Structural Analysis

Summary

We describe an integrated workflow for chemical cross-linking of proteins with mass spectrometry to study biological complexes in vivo. The protein interaction reporter (PIR) cross-linker presents features that enable the cross-linking of living cells with no prior protein isolation needed, providing information on protein conformations and protein-protein interactions.

Abstract

Chemical cross-linking of proteins with mass spectrometry (XL-MS) has increasingly become a powerful technique when studying protein structures and complexes. This approach is based on the reactivity of cross-linkers to specific protein sites - usually primary amines, including side chains of lysine residues and protein N-termini which yields information on protein-protein interactions and protein conformations. Information provided by XL-MS is complementary to that from other structural methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. Here, we describe a protocol for in-house synthesis and use of a peptide-based cross-linker with optimized features for interactome studies of complex biological samples. These features comprise the protein interaction reporter (PIR) technology, MS-cleavable bonds, and an affinity tag, which ultimately facilitate the identification of cross-linked peptide pairs. The membrane permeability enables the cross-linking of living cells, tissues, and isolated organelles (e.g., nuclei and mitochondria), providing valuable structural and interaction data on proteins as they exist in their native environment. Moreover, quantitative XL-MS can be utilized for comparative interactome studies, providing information on protein conformational and interaction changes between varying biological states.

Introduction

Biological processes are driven by multiple and complex mechanisms, with different molecules - nucleic acids, proteins, carbohydrates, lipids, etc. - playing key roles in each step. When studying proteins, several approaches can be used, but the ultimate objective is to understand how proteins with different domains and regulatory regions are structurally organized and functioning in a crowded cellular environment^1,2. Besides structural information, assessing protein interactors and complexes is essential for the understanding of cellular mechanisms.

Chemical cross-linking of protei....

Protocol

NOTE: All materials, equipment, and software used here are described in Table of Materials.

1. Synthesis of BDP (Figure 3)

NOTE: The BDP-NHP cross-linker was synthetized using a CEM Liberty Lite peptide synthesizer, following the manufacturer's instructions. This protocol is based on a previous publication⁷.

1. Prepare the following reagents.
   1. Weigh out 0.63 g of Rink Amide resin (100-200 mesh) for 0.05 mmol.
   2. Weigh out 1.73 g of aspartate (Fmoc-Asp(OtBu)-OH) and dissolve it in 21 mL of DMF.
   3. Weigh out 23 g of succin....

Discussion

XL-MS can provide information on protein structure and conformation (intra-links), protein-protein interaction and complex assembly (inter-links), and protein quantitation and solvent exposure (dead-ends). One limitation of the technique when used in vivo for complex samples is the low-resolution structural results on protein conformation and interactome. Thus, protein structures present in the PDB are useful for the modelling of structures and for the visualization of cross-links, which is automatically done by.......

Materials

Name	Company	Catalog Number	Comments
Materials
Acetonitrile	Fisher Scientific	A955	LC–MS grade
Ammonium bicarbonate	Sigma-Aldrich	A6141	NH4HCO3
Ammonium hydroxide	Sigma-Aldrich	221228	NH4OH, ACS reagent, 28.0–30.0% NH3 basis
Calcium chloride	Sigma-Aldrich	AC42352	CaCl2
Dichloromethane	Fisher Scientific	AC610300010	DCM, 99,9% (wt/wt)
Dichloromethane	Fisher Scientific	AC610300010	DCM; 99.9% (wt/wt)
Diethyl ether 99.5% (wt/wt)	Fisher Scientific	AC364330010
Diisopropylcarbodiimide	Sigma-Aldrich	D125407	DIC
Dimethyl sulfoxide	Sigma-Aldrich	276855	DMSO
Dimethylformamide	Fisher Scientific	D119	DMF
DMEM	Fisher Scientific	11-965-118	Cell culture medium
EDTA 99% (wt/wt)	Fisher Scientific	AC118432500
Ethyl cyano(hydroxyimino)acetate	Sigma-Aldrich	8510860100	Oxyma
Fetal bovine serum (FBS)	Atlas Biologicals	FP-0500-A
Fmoc-Asp(OtBu)-OH	Millipore Sigma	8520370005
Fmoc-Gly-Wang resin	Bachem	D-1745	100–200 mesh
Fmoc-L-Lys(biotin)-OH	P3 BioSystems	41084
Fmoc-Lys(Fmoc)-OH	Millipore Sigma	8520410025
Fmoc-Pro-OH	Millipore Sigma	8520170025
Formic acid	Fisher Scientific	A117	FA, 99.5+% (vol/vol), LC–MS grade
HeLa cells	ATCC	CCL-2	Epithelial from human cervix
Iodoacetamide	Sigma-Aldrich	I1149	IAA
Magnesium chloride	Sigma-Aldrich	M8266	MgCl2
Methanol	Fisher Scientific	A456	LC–MS grade
Monomeric Avidin UltraLink Resin	Pierce Biotechnology	53146
N-hydroxyphthalimide	Sigma-Aldrich	H53704	NHP
Nitrogen (g) (99.998%)	Praxair	NI 4.8-T
PBS	Fisher Scientific	SH30256LS
PBS with calcium and magnesium	Fisher Scientific	SH30264FS
Penicillin–streptomycin (10,000 U/mL)	Fisher Scientific	SV30010
Piperidine	Sigma-Aldrich	411027	99.5% (vol/vol)
Poly-Prep chromatography columns	Bio-Rad	7311550	Disposable polypropylene columns
Potassium chloride	Sigma-Aldrich	P9541	KCl
Potassium phosphate, monobasic	Sigma-Aldrich	P9791
Pyridine	Millipore Sigma	MPX20127	DriSolv, anhydrous septum-sealed bottle
ReproSil-Pur, 5 micron, 120 Å	Dr. Maisch	r15.8e
Sep-Pak Vac 3cc (500mg) C18 cartridges	Waters	186004619	reversed-phase C18 desalting column
Sodium chloride	Sigma-Aldrich	S 9888
Sodium hydroxide	Sigma-Aldrich	221465
Sodium phosphate, dibasic	Sigma-Aldrich	AC20651
Sodium phosphate, monobasic (NaH2PO4; Sigma-Aldrich, cat. no. S9638)	Sigma-Aldrich	S9638
Succinic anhydride	Sigma-Aldrich	239690
TFA acid	Fisher Scientific	LS121	LC–MS grade
Trifluoroacetic acid	Fisher Scientific	A116	TFA acid, Optima LC–MS grade
Trifluoroacetic anhydride	Sigma-Aldrich	106232	TFA anhydride
Tris(2-carboxyethyl)phosphine hydrochloride	Fisher Scientific	20491	TCEP-HCl
Trypsin	Promega	V5113	sequencing grade, modified, frozen
Urea	Sigma-Aldrich	U5378
Water	Fisher Scientific	W6	LC–MS grade
Water with 0.1% (vol/vol) TFA acid	Fisher Scientific	LS119	LC–MS grade
Equipments
1.5-mL LC autosampler vial	Thermo Scientific	MSCERT4000-39TR
Analytical/preparative HPLC system	Agilent Technologies	G1311C
CEM Liberty Lite peptide synthesizer	CEM		Automated Microwave Peptide Synthesizer
Conical centrifuge tubes	Fisher Scientific	14-959-53A and 12-565-270	15 and 50 mL
Extraction manifold	Waters	WAT200608	20 position, 16 × 75-mm tubes
LC autosampler vial caps	Fisher Scientific	13-622-289
LC autosampler vials	Fisher Scientific	03-377-299
Microcentrifuge	Eppendorf	22620444
Microcentrifuge tubes	Fisher Scientific	02-681-320	1.5 mL
pH paper	Fisher Scientific	13-640-5070
Poly-Prep chromatography columns	Bio-Rad	7311550
Q Exactive Plus	Thermo Scientific	IQLAAEGAAPFALGMBDK	High resolution mass spectrometer
SCX column	Phenomenex	00G-4398-N0	Luna column
Sep-Pak Vac C18 cartridge, 3cc/500mg	Waters	186004619
Shaker with 15- and 1.5-mL sample blocks	Eppendorf	5355	ThermoMixer R
Thermal mixer	Sigma-Aldrich	T1317	Eppendorf ThermoMixer compact
Ultimate 3000	Thermo Scientific	ULTIM3000RSLCNANO	nano-LC system
Ultrasonic processor	Cole-Palmer	EW-04714-50
Vacuum centrifuge	SP Scientific	EZ-2
Name of Software
ReAdW (https://sourceforge.net/projects/sashimi/files/ReAdW%20%28Xcalibur%20converter%29/)
Mango (https://github.com/jpm369/mango)
Comet 2018.01 or later (http://comet-ms.sourceforge.net/)
XLinkProphet (https://github.com/brucelab/xlinkprophet)
Perl v.5.24.0+ (https://www.perl.org/get.html)
All required software can be run on a standard personal computer equipped with a Linux operating system and at least 4 GB of RAM.