Mar 30, 2026
Version 2
scSP: Single Cell Spatial Proteomics LC-MS/MS Analysis of LCM-isolated single-cell mouse brain tissue V.2
- 1California Institute of Technology
Protocol Citation: Marion Pang, Sayan Dutta 2026. scSP: Single Cell Spatial Proteomics LC-MS/MS Analysis of LCM-isolated single-cell mouse brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwb2pdvmk/v2Version created by Marion Pang
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: February 07, 2025
Last Modified: March 30, 2026
Protocol Integer ID: 119747
Keywords: bottom-up mass spectrometry, LCM, proteomics, mouse brain, single cell spatial proteomics lc, cell mouse brain tissue this protocol, cell mouse brain tissue, mouse brain tissue, cell tissue sample, mass spectrometry analysis, cell tissue samples for bottom, ms analysis of lcm, procedure for scsp, tissue sample
Abstract
This protocol outlines the procedure for scSP: Single Cell Spatial Proteomics LC-MS/MS Analysis of LCM-isolated single-cell mouse brain tissue. It discusses the specifics of laser-capture micro-dissection from mouse brain tissue using a Leica LMD7000/LMD7 with Leica Laser Microdissection Software Version V8.4, followed by the procedure for preparing single-cell tissue samples for bottom-up mass spectrometry analysis. Lastly, it discusses the configuration for LC-MS/MS analysis.
Guidelines
This protocol was optimized for single cell tissues isolated from a 30um fixed mouse brain section. Laser parameters may need to be adjusted depending on the sample to be dissected.
Materials
Equipment:
Leica LMD7000, Laser Microdissection Software Version V8.4
Leica LMD7, Laser Microdissection Software
QuantStudioTM Real-Time PCR Machine
Centrifuge
Vanquish Neo UHPLC, Thermo Scientific
Orbitrap Exploris 480 mass spectrometer, Thermo Scientific
Nanospray Flex ion source (Thermo Scientific)
Protocol materials
Triethylammonium BicarbonateThermo ScientificCatalog #90114
n-Dodecyl-beta-Maltoside DetergentThermo FisherCatalog #89902
Trypsin, Mass Spectrometry GradePromegaCatalog #V5280
Lys-C, Mass Spectrometry GradeWako ChemicalsCatalog #125-05061
Acetonitrile, LC-MS gradeThermo Fisher ScientificCatalog #51101
Formic acid, LC-MS gradeThermo Fisher ScientificCatalog #28905
n-Dodecyl-beta-MaltosideThermo ScientificCatalog #89903
Triethylammonium Bicarbonate Thermo ScientificCatalog #90114
Safety warnings
This protocol involves use of a 349 nm adjustable pulse laser (max 120uJ). Ensure the safety guard is attached and do not look directly into the laser while it is on.
Wear proper PPE while handling chemicals.
Ethics statement
Before beginning the procedure, ensure that all experimental steps have been reviewed and approved under the appropriate institutional protocols, and that all personnel involved are properly trained and compliant with institutional regulations.
Before start
Prepare lysis buffer fresh on the day.
Samples should be stored on ice during transport.
Maintenance and cleaning of the LC and MS systems are crucial for collecting good quality data.
Sample Isolation
Following tissue processing and staining processes (optional), tissue sections are mounted on a PET membrane metal frame slide.
Equipment
PET membrane (with metal frame), RNAse free
NAME
Slide
TYPE
Leica
BRAND
#76463-322
SKU
LINK
Proteomics (HPLC, Mass Spec) - no softeners, little autofluorescence
SPECIFICATIONS
00:01:00
Regions of interest were excised from mounted tissues using a gravity-driven collection system by a Leica
LMD7000 or a Leica LMD7 (Leica Laser Microdissection Software Version V8.4). The laser settings are defined as below.
| A | B | C | |
| Magnification | 20X | 40X | |
| Power | 55 | 24 | |
| Aperture | 3 | 1 | |
| Speed | 3 | 12 | |
| Specimen balance | 15 | 12 | |
| Line spacing for Draw + Scan | 15 | 19 | |
| Head current | 100% | 100% | |
| Pulse frequency | 1510 | 1046 | |
| Offset | 115 | 180 |
Leica LMD7000 Laser Settings for 20x and 40x magnification.
| A | B | |
| Magnification | 40X | |
| Power | 45 | |
| Aperture | 1 | |
| Speed | 25 | |
| Specimen balance | 15 | |
| Line spacing for Draw + Scan | 19 | |
| Head current | 100% | |
| Pulse frequency | 519 | |
| Offset | 180 |
Leica LMD7 Laser Settings for 20x and 40x magnification.
Note
Settings were optimized on 30um fixed mouse brain sections. Parameters should be optimized based on sample type.
Sample Storage
Collect the dissected tissue in the cap of a 0.65 mL Low Binding Tube containing 10 µL of lysis buffer consisting of 50 millimolar (mM) Triethylammonium Bicarbonate Thermo ScientificCatalog #90114 and 0.2 Mass / % volume n-Dodecyl-beta-MaltosideThermo ScientificCatalog #89903 .
Equipment
Sorenson SafeSeal Low Binding Tube
NAME
Tube
TYPE
Sorenson
BRAND
11300
SKU
0.65mL
SPECIFICATIONS
Note
It is important to ensure that the buffer covers as much surface area of the cap as possible, to maximize odds of sample recovery.
Other protein low binding tubes may be used, but must first be verified to fit in the sample holder of the LMD7000.
The use of an anti-static gun may be beneficial for higher efficiency drop rates.
Visually verify that the dissected tissue has landed on the cap.
Vortex the tube, keeping it held upside down for 30 seconds.
Centrifuge the tubes at 13000 x g, 25°C, 00:01:00 .
Store the samples at -80 °C until further processing.
Lysis
Retrieve samples from the -80 °C freezer and allow to thaw till Room temperature .
For best recovery of samples, add 5 µL of lysis buffer (0.2 Mass / % volume n-Dodecyl-beta-Maltoside DetergentThermo FisherCatalog #89902 in 50 millimolar (mM) Triethylammonium BicarbonateThermo ScientificCatalog #90114 ) to each tube, then votex in an inverted position for 30s.
Centrifuge at 13000 x g, Room temperature, 00:01:00 .
Transfer samples into a LoBind 384 well PCR plate and incubate at 0 rpm, 70°C, 01:00:00 in a PCR machine, with the lid set to 85 °C .
Equipment
LoBind 384 well PCR plate
NAME
Plate
TYPE
Eppendorf
BRAND
0030129547
SKU
Note
We have found that incubation using a tight-press PCR machine with a heated lid can help to increase the efficiency of sample lysis and protein denaturation.
Digestion
Following lysis, centrifuge the plate 1000 x g, Room temperature, 00:01:00 and allow the plate to cool to room tempature.
Add1 µL of protease digestion mix consisting of 4 ng Trypsin, Mass Spectrometry GradePromegaCatalog #V5280 and 4 ng Lys-C, Mass Spectrometry GradeWako ChemicalsCatalog #125-05061 to each well.
Incubate the plate at 37 °C Overnight .
Centrifuge the plate at 1000 x g, Room temperature, 00:01:00 and quench digestion with 0.5 µL aqueous buffer comprising of 2 % volume Acetonitrile, LC-MS gradeThermo Fisher ScientificCatalog #51101 and 4 % volume Formic acid, LC-MS gradeThermo Fisher ScientificCatalog #28905 .
The peptides are now ready for LC-MS/MS analysis, and can be stored in -80 °C until further processing.
The steps below detail LC-MS/MS acquisition on two separate LC-MS systems.
Vanquish Neo-Orbitrap Exploris 4807 steps
Using a Vanquish Neo-Orbitrap Exploris 480 system
Peptides are separated on an Aurora Ultimate UHPLC Column (25cm by 75µm), with column temperature maintained at 50 °C .
5m
5 µL of sample from each well is loaded onto the column.
The separation gradient is configured with a flow rate of 0.22 µL/min and an analytical duration of
01:00:00 . Further information of gradient conditions can be found in the supplementary information (Table S1 and S2).
1h
The LC system (Vanquish Neo UHPLC, Thermo Scientific) was coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) with a Nanospray Flex ion source (Thermo Scientific).
The LC-MS was configured for two types of data acquisition methods, which are detailed below.
Data-dependent acquisition (DDA) 2 steps
Data-dependent acquisition (DDA) was carried out in positive ion mode using a positive ion voltage of 1600 V while maintaining the ion transfer tube at a temperature of 300 °C . The maximum injection time was set to auto, and the normalized AGC target was set to 300%. Precursor ions with charges ranging from +2 to +6 were selectively targeted for fragmentation using a minimum intensity threshold of 5e3. Dynamic exclusion was set to exclude after one acquisition, with a 45-second exclusion duration and 10 ppm mass tolerance. MS2 scans were acquired in the Orbitrap at 60000 resolutions with an isolation window of 1.6 m/z, HCD collision energy set at 28%, and an auto-adjusted maximum injection time. The normalized AGC target was set at 200%. Xcalibur software (Thermo Scientific) was used for method implementation and data acquisition.
Xcalibur software (Thermo Scientific) was used for method implementation and data acquisition.
Protocol references
Matzinger, M., Müller, E., Dürnberger, G., Pichler, P. & Mechtler, K. Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics. Anal. Chem. 95, 4435–4445 (2023).