Mar 28, 2025
Cross-Linking and Strong Cation Exchange (SCX) Fractionation
- Miguel A. Gonzalez-Lozano1,
- Harper JW2
- 1Harvard Medical School;
- 2harvard university
Protocol Citation: Miguel A. Gonzalez-Lozano, Harper JW 2025. Cross-Linking and Strong Cation Exchange (SCX) Fractionation. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5q2og47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2024
Last Modified: March 28, 2025
Protocol Integer ID: 110306
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 000282, 025160
Abstract
This is a method for performing Lys-Lys cross-linking on a purified organelle sample, in this case EEA1-positive early endosomes. The crosslinker used is DSSO ((Bis(2,5-dioxopyrrolidin-1-yl)
3,3′-sulfinyldipropionate)). This cross-linker has a mass spec cleavable sulfoxide, which facilitates identification of individual cross-linked peptides.
Materials
disuccinimidyl sulfoxide (DSSO; Thermo Scientific, A33545)
2-chloroacetamide (Sigma-Aldrich, C0267)
trypsin (Promega, V511C)
Lys-C (Wako Chemicals, 129-02541)
Sep-Pak C8 50mg Cartridge (Waters, WAT054965)
EPPS (3-[4-(2-Hydroxyethyl)-1-piperazine]propanesulfonic acid) (Thermo Scientific, J61296AE)
dithiothreitol (DTT)
Empore SPE Disks C18 to generate stage tips(Sigma Millipore, 66883-U)
PolyLC PolySulfoethyl A column (3 μm particle size, 2.1 mm inner diameter, and 100 mm length) (#102SE03--)
Acetonitrile
Formic acid
Lys-C (Wako Chemicals, 129-02541)
Organelle preparations
Organelle preparations
Samples for cross-linking (e.g. individual organelles) are isolated using dedicated protocols such as https://doi.org/10.17504/protocols.io.ewov14pjyvr2/v2 for isolation of EEA1-positive early endosomes
Protein cross-linking
Protein cross-linking
Resuspend freshly prepared purified organelle pellets in KPBS (25 mM KCl, 100 mM potassium phosphate, 150 mM NaCl, pH 7.2) to a protein concentration of ~1μg/uL.
Cross-link the proteins by adding 1 mM DSSO (Bis(2,5-dioxopyrrolidin-1-yl) 3,3′-sulfinyldipropionate) and incubate at room temperature for 40 minutes.
Quench the cross-linking reaction by adding 50 mM Tris buffer (pH 7.5) and incubate at room temperature for 30 minutes.
Alternatively, other cross-linker reagents could be use. For DHSO/DMTMM, 8 mM DHSO (3,3'-sulfinyldi(propanehydrazide)) and 16mM DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) at 37°C for 90 min.
Proceed directly with protein precipitation. Samples can be digested using S-trap sample preparation following mini-spin column digestion protocol as provided by the manufacturer (Protifi, C02-mini-80).
Protein digestion
Protein digestion
Denature the cross-linked samples by adding 8 M urea.
Reduce the proteins by adding 5 mM dithiothreitol (DTT) and incubate for 30 minutes at 37°C.
Alkylate the reduced proteins with 40 mM chloroacetamide for 30 minutes at room temperature.
Digest the cross-linked proteins with Lys-C enzyme at a ratio of 1:75 (enzyme) and incubate at 37°C overnight.
Dilute the urea concentration of the sample to 2 M using 50 mM EPPS buffer (pH 8.0).
Further digest the proteins by adding trypsin at a ratio of 1:100 (enzyme) and incubate at 37°C for 6 hours.
Desalt the peptides using Sep-Pak solid-phase extraction column.
Dry the desalted peptides in a SpeedVac concentrator. Time will depend on the vacuum strength.
Peptide separation by SCX
Peptide separation by SCX
Fractionate the dried peptides by strong cation exchange (SCX) chromatography.
A 70-minute linear gradient at flow rate 0.18 mL/min was used as follows:
- 0% to 8% of mobile phase (0.5 M NaCl in 20% ACN, 0.05% formic acid) over 14 minutes.
- Increase to 20% at 28 minutes.
- Increase to 40% at 48 minutes.
- Increase to 90% at 68 minutes.
Collect fractions every 30 seconds starting at 35 minutes for 10 minutes, and then every minute thereafter.
Dry the collected fractions in a SpeedVac concentrator.
Desalt the dried fractions using StageTip.
Reconstitute fractions in 5% acetonitrile (ACN) and 5% formic acid.
Analyze the reconstituted fractions by liquid chromatography-tandem mass spectrometry (LC-MS/MS).