Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Summary

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Abstract

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Introduction

Our understanding of cell differentiation and the genesis of tissues and organs is the result of decades of elaborate targeted screens of genes and their products. Increasing our knowledge of all the biomolecules and their quantities during important cellular events would help unravel molecular mechanisms that control the spatial and temporal patterning of the vertebrate body plan. Technologies enabling molecular amplification and sequencing are now able to routinely report on large numbers of genes and transcripts, supporting hypothesis-driven studies in basic biological and translational research. To understand developing systems, a complex relationship between tran....

Protocol

All protocols ensuring the humane maintenance and handling of Xenopus laevis adult frogs were approved by the Institutional Animal Care and Use Committee at the University of Maryland, College Park (Approval numbers R-DEC-17-57 and R-FEB-21-07).

1. Prepare the solutions

1. For embryology
   1. Prepare 1x, 0.5x, and 0.2x Steinberg's solution (SS), then autoclave them (120 °C for 20 min) to sterility following standard protocols¹².
   2. Prepare 3% (w/v) Ficoll in sterilized 1x SS following standard protocols¹².
   3. For dejellying, fr....

Results

This protocol enabled the study of proteins in single cells and their lineages as they establish tissues in X. laevis embryos. Figure 1 illustrates one such application of the approach to study proteins in neural-tissue-fated cells and the newly induced neural ectoderm in the embryo. As shown in Figure 1A, the bioanalytical workflow integrated traditional tools of cell and developmental biology to identify, inject/aspirate cells, and collect specimens. .......

Discussion

This protocol enables the characterization of protein expression in identified cell lineages in embryos of the Xenopus species. Stemming from HRMS, the methodology combines exquisite specificity in molecular identification, capability for multi-protein detection without molecular probes (usually hundreds to thousands of different proteins), and a capability for quantification. Adaptability to classical tools and workflows in cell and developmental (neuro)biology expand HRMS proteomics to exciting applications, i.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile (LC-MS-grade)	Fisher Scientific	A955
Agarose	ThermoFisher Scientific	R0492
Ammonium bicarbonate	Fisher Scientific	A643-500
Analytical Column	Thermo Scientific	164941
Analytical microbalance	Mettler-Toledo	XSE105DU
Automatic peptide fractionation platform	Agilent	1260 Infinity II
Borosilicate Capillaries	Sutter Instruments Co.	B100-50-10
Borosilicate Capillaries (for making Emmitters)	Sutter Instruments	B100-75-10
C18 spin columns (for desalting)	ThermoFisher Scientific	89870
Camera ro monitor electrospray	Edmund Optics Inc.	EO-2018C
Combretastatin A4	Millipore Sigma	C7744
Commercial CESI system	AB SCIEX	CESI
(Cyclohexylamino)-1-propanesulfonic acid (CAPS)	VWR	97061-492
Cytochalasin D	Millipore Sigma	C8273
Dextran, Alexa Fluor 488; 10,000 MW, Anionic, Fixable	ThermoFisher Scientific	D22910
Diothiothreitol	Fisher Scientific	FERR0861
Dumont #5 Forceps	Fine Science Tools	11252-30
EDTA	Fisher Scientific	AAJ62786AP
Epifluorescence light source	Lumencore	AURA III
Eppendorf LoBing microcentrifuge  tubes: protein	Fisher Scientific	13-698-793
Formic acid (LC-MS-grade)	Fisher Scientific	A117-50
Freezer (-20 °C)	Fisher Scientific	97-926-1
Freezer (-80 °C)	Thermo Scientific	TSX40086A
Fused silica capillary	Molex	1088150596
Heat Block	Benchmark	BSH300
High pressure liquid Chromatography System	ThermoFisher Scientific	Dionex Ultimate 3000 RSLC nanosystem
High voltage power supply	Spellman	CZE1000R
High-resolution Mass Spectrometer	ThermoFisher Scientific	Orbitrap Fusion Lumos Tribrid Mass Spectrometer
HPLC caps	Thermo Scientific	C4013-40A
HPLC Vials	Thermo Scientific	C4013-11
Illuminator e.g. Goosenecks	Nikon	C-FLED2
Ingenuity Pathway Analysis	Qiagen
Iodoacetamide	Fisher Scientific	AC122275000
Methanol (LC-MS-grade)	Fisher Scientific	A456
Methanol (LC-MS-grade)	Fisher Scientific	A456-4
Microcapillary puller	Suttor Instruments	P-2000
Microinjector	Warner Instrument, Handem, CT	PLI-100A
Micropippette puller	Sutter Instruments Co.	P-1000
MS data analysis software, commercial	ProteomeDiscoverer
MS data analysis software, opensource	MaxQuant
non-idet 40 substitute	Millipore Sigma	11754599001
Petri dish 60 mm and 80 mm	Fisher Scientific	S08184
Pierce 10 µL bed Zip-tips (for desalting)	ThermoFisher Scientific	87782
Pierce bicinchoninic acid protein assay kit	ThermoFisher Scientific	23225
Pierce quantitative colorimetric peptide assay	ThermoFisher Scientific	23275
Pierce Trypsin Protease (MS Grade)	Fisher Scientific	PI90058
Protein LoBind vials	Eppendorf	0030108434 , 0030108442
Refrigerated Centrifuge	Eppendorf	5430R
Refrigerated Incubator	Thermo Scientific	PR505755R/3721
sodium isethionate	Millipore Sigma	220078
sodium pyrophosphate	Sigma Aldrich	221368-100G
Stainless steel BGE vial	Custom-Built
Stainless steel sample vials	Custom-Built
Stereomicroscope (objective 10x)	Nikon	SMZ 1270, SZX18
Sucrose	VWR	97063-790
Syringe pumps (2)	Harvard Apparatus	704506
Syringes (gas-tight): 500–1000 µL	Hamilton	1750TTL
Transfer pipettes (Plastic, disposable)	Fisher Scientific	13-711-7M
Trap Column	Thermo Scientific	164750
Tris-HCl (1 M solution)	Fisher Scientific	AAJ22638AP
Vacuum concentrator capable of operation at 4–10 °C	Labconco	7310022
Vortex-mixer	Benchmark	BS-VM-1000
Water (LC-MS-grade)	Fisher Scientific	W6
Water (LC-MS-grade)	Fisher Scientific	W6
XYZ translation stage	Thorlabs	PT3
XYZ translation stage	Custom-Built