Protein extraction of zebrafish and fathead minnow embryos

This protocol describes an optimized protein extraction method for proteomic analyses for zebrafish and fathead minnow embryo's aged ~96 hpf/dpf (species dependent), and wouldn't have been possible without the following papers

References:

Masuda, T., Tomita, M., Ishihama, Y., 2008. Phase Transfer Surfactant-Aided Trypsin Digestion for Membrane Proteome Analysis. J. Proteome Res. 7, 731–740. https://doi.org/10.1021/pr700658q

Lin Y, Lin H, Liu Z, Wang K, Yan Y. Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics. J Sep Sci. 2014; 37(22): 3321-9. doi: 10.1002/jssc.201400569

Rappsilber, J., Ishihama, Y., Mann, M., 2003. Stop And Go Extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670. https://doi.org/10.1021/ac026117i

Wiśniewski, J.R., 2016. Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols. Analytical Chemistry 88, 5438–5443. https://doi.org/10.1021/acs.analchem.6b00859

Wiśniewski, J.R., Zougman, A., Nagaraj, N., Mann, M., 2009. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362. https://doi.org/10.1038/nmeth.1322

Before starting

• Prepare stock solutions
• Samples can be lysed and quantified in advance and stored at -20/-80 C, therefore sample preparation and peptide digestion are separated into two categories. If samples are prepared in advance, then only PBS wash solution, lysis buffer and 10 % SDS stock need to be made in advance.
• Defrost and store samples on ice
• Pre-heat thermo-shaker (95 °C)

A	B	C
Item	Retailer	Catalogue
Phosphate buffered saline (PBS)	Sigma-Aldrich	P4417
HPLC water	Sigma-Aldrich	34877
Trizma hydrochloride (Tris-HCL)	Sigma-Aldrich	T3253
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	71726
cOmplete Ultra tablets (Protease inhibitor cocktail)	Sigma-Aldrich	5892970001
Methanol	Sigma-Aldrich	A456-4
Ammonium acetate	Sigma-Aldrich	A7262
Acetone	Sigma-Aldrich	179124
Pierce BCA protein assay kit	Thermo Fisher Scientific	23225
Bovine serum albumin (BSA; 1mg/mL)	Fisher Scientific	AAJ60205AD
Amicon Ultra-0.5 Centrifugal Filter Unit (3K)	Sigma-Aldrich	UFC5003BK
Urea	Sigma-Aldrich	U5128
Ammonium Bicarbonate Buffer (abc)	Sigma-Aldrich	S2454
Dithiothreitol (DTT)	Sigma-Aldrich	646563
Iodoacetamide (IAA)	Sigma-Aldrich	I1149
Sodium deoxycholate (SDC)	Sigma-Aldrich	D6750
Sodium chloride solution	Sigma-Aldrich	S5150
Trypsin (frozen)	Promega	V5113
Trifluoroacetic acid (TFA)	ProteoChem	LC6203
Pierce C18 tips	Thermo Fisher Scientific	87782
Acetonitrile	Fisher Scientific	A955
Formic acid	Sigma-Aldrich	F0507

Stock solutions

PBS wash solution

• 1 tablet/100 mL of water

Lysis buffer

• 0.1 M Tris-HCL (pH 7.8)
• 0.05 M DTT
• cOmplete Ultra tablets (Protease inhibitor cocktail) [1 tablet/10 mL buffer]

10 % SDS stock solution (w/v) [HPLC water]

UA buffer

• 2 M Urea (pH 8.5; 0.1 M Tris-HCL)

Digestion buffer (DB)

• ABC buffer (50 mM)

DTT buffer

45 1 M stock to 955 HPLC water

IAA stock (1M)

• 189 mg/1 mL ABC buffer

Sodium deoxycholate (SDC), 10 % (w/v)

Elution buffer

• 70 % NaCL [1 M] + 30 % methanol
• ABC buffer (sodium bicarbonate)

5 % TFA (v/v)

Equipment

• Micro-centrifuge
• Tissue homogenizer (e.g. Scilogex D160 homogenizer + saw tooth probe)
• Sonicating probe (Misonix sonicator; ultrasonic liquid processor with micro-tip)
• ThermoMixer C eppendorf + smartblock and/or heating block
• Speedvac