Literature DB >> 29876449

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data.

Balkis Eddhif¹, Nadia Guignard², Yann Batonneau², Jonathan Clarhaut^3,4, Sébastien Papot³, Claude Geffroy-Rodier¹, Pauline Poinot¹.

Abstract

The data presented here are related to the research paper entitled "Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses" (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

Entities: Chemical Disease Gene Species

Year: 2018 PMID： 29876449 PMCID： PMC5988388 DOI： 10.1016/j.dib.2018.01.095

Source DB: PubMed Journal: Data Brief ISSN： 2352-3409

Specifications Table Value of the data Data show a comprehensive evaluation of protein conformational changes throughout TCA precipitation and one single step purification with various solvents. Data highlight the efficiency of ethanol/HCl purification for TCA-precipitated proteins. Ethanol/HCl represents a quick and inexpensive purification agent for proteomics studies. Presence and variability of proteins are potential values to determine which purification method must be used for proteomics investigation.

Data

TCA precipitation is one of the most common and robust technique required for protein analyses [5], [6], [7]. However it leads to molten globule states which hamper the solubilization of proteins in aqueous buffers for mass spectrometry analysis.

Comparison of washing agents on standard solutions

A standard solution of HSA, cellulase (exoglucanases and endoglucanases mixture), lysozyme and ribonuclease A, 35 µg mL−1 each, was prepared in high purified water. Prot eins were precipitated with 25% (w/v) trichloroacetic acid (TCA) (final concentration). The clean-up of protein pellet was performed following three different approaches: ethanol/HCl (1.25 M; 3.8%), acetone/HCl (0.06 M; 0.2%); acetone/HCl (1.25 M; 3.8%) (Fig. 1).

Fig. 1

Standard proteins quantification by Bradford assay and silver-staining on electrophoresis gel. The thin line bars represent standard deviations at the top of the Bradford histogram. For both methods, histograms were constructed from the mean value of three independent assays.

Extraction and purification of endogenous proteins from complex sample matrices

See Fig. 2.

Fig. 2

One-dimensional gel electrophoresis of complex matrices (biofilm, soil and mouse liver) after purification following the designed approach versus published protocols on complex matrices. The gel was stained with silver nitrate.

Effects of successive ethanol/HCl washings on proteins recoveries

10 mg of biofilm samples were spiked with the standard solution of HSA, exoglucanase 1 from the mix of cellulase, lysozyme, and ribonuclease A (Rnase). Proteins final concentration was 1 µg mg−1 of matrix to enable HRMS detection of the proteins after the whole process. The mixture was vortexed and left during 24 h at room temperature to favor proteins adsorption on the matrix. After extraction following the published protocol of Huang et al. [3], protein pellets were subjected to one, two or three ethanol/HCl washing(s). They were then dissolved in 50 mM of ammonium bicarbonate containing 10 mM of Tris (pH 8.5), diluted in a ratio of 1:3 using the same buffer and subjected to trypsin digestion. Experiments were performed in triplicate. Fig. 3 gives the mean protein recoveries following the designed approach (Ethanol/HCl) on biofilm matrix after multiple washing steps.

Fig. 3

Proteins recoveries following the designed approach on biofilm sample. The thin line bars represent standard deviations at the top of each column. Each bar shows mean±s.e.m. from three independent purification assays. Protein recoveries in Tris buffer were determined by UPLC/HRMS in a full scan mode with a resolution of 70.000 and mass range of 200–3000 m/z.

Understanding the effect of ethanol/HCl on proteins conformation

Spectrofluorometry

To get insights into the role of ethanol/HCl on proteins solubility, their conformational changes were comprehensively investigated, as an extension of the results reported in Ref. [1]. These measures were performed at each purification stage with two spectroscopic techniques: spectrofluorometry and Raman. Fig. 4, Fig. 5, Fig. 6 represent the fluorescence emission spectra of lysozyme, HSA and Rnase after TCA precipitation and washing steps (ethanol/HCl, ethanol or acetone).

Fig. 4

Fig. 5

Emission spectra of HSA (λexc = 400 nm) at different purification steps. Native HSA (grey spectrum); HSA-TCA (orange spectrum); HSA-ethanol/HCl (green spectrum); HSA-ethanol (purple spectrum); HSA-acetone (blue spectrum).

Fig. 6

Emission spectra of RNASE (λexc = 400 nm) at different purification steps. Native Rnase (grey spectrum); Rnase-TCA (orange spectrum); Rnase-ethanol/HCl (green spectrum); Rnase-ethanol (purple spectrum); Rnase-acetone (blue spectrum).

Emission spectra of lysozyme (λexc = 400 nm) at different purification steps. Native lysozyme (grey spectrum); Lysozyme-TCA (orange spectrum); Lysozyme-ethanol/HCl (green spectrum); Lysozyme-ethanol (purple spectrum); Lysozyme-acetone (blue spectrum). Emission spectra of HSA (λexc = 400 nm) at different purification steps. Native HSA (grey spectrum); HSA-TCA (orange spectrum); HSA-ethanol/HCl (green spectrum); HSA-ethanol (purple spectrum); HSA-acetone (blue spectrum). Emission spectra of RNASE (λexc = 400 nm) at different purification steps. Native Rnase (grey spectrum); Rnase-TCA (orange spectrum); Rnase-ethanol/HCl (green spectrum); Rnase-ethanol (purple spectrum); Rnase-acetone (blue spectrum).

Raman microspectroscopy

Raman spectrum for Rnase, is presented in Fig. 7. Spectra and curve fitting of the amide I band of proteins corresponding to lysozyme and HSA are presented in Fig. 5, Fig. 6 in Ref. [1], respectively (Fig. 8, Fig. 9, Fig. 10, Fig. 11).

Fig. 7

Fig. 8

Difference spectra (experimental - fitting curve) after analysis of the amide I Raman bands of lysozyme at different purification steps (Fig. 5, [1]). a. Native lysozyme (blue); b. Lysozyme-TCA (red); c. Lysozyme-ethanol/HCl (black).

Fig. 9

Difference spectra (experimental – fitting curve) after analysis of the amide I Raman bands of HSA at different purification steps (Fig. 6, [1]). a. Native HSA (blue); b. HSA-TCA (red); c. HSA-ethanol/HCl (black).

Fig. 10

Relative integrated intensities of lysozyme amide I contribution from peak #6 assigned to unordered structures (uo), peak#7 (ordered α helices, ho), peak#8 (unordered α helices and β sheets, hu+sh), and peak #9 (turns, tu) as obtained after profile fitting of amide I region of the Raman spectra (Fig. 5, Ref. [1]). Values on top of each bar correspond to the Raman shift on which the contribution peak was centred at the end of the fitting.

Fig. 11

Relative integrated intensities of HSA amide I contribution from peak #1 assigned to unordered structures (uo), peak#2 (ordered α helices, ho), peak#3 (unordered α helices and β sheets, hu+sh), and peak #4 (turns, tu) as obtained after profile fitting of amide I region of the Raman spectra shown in Fig. 6[1]. Values on top of each bar correspond to the Raman shift on which the contribution peak was centred at the end of the fitting.

Raman spectra of Rnase at different purification steps (range 1200–1800 cm−1). a. Native Rnase (blue spectrum); b. Rnase-TCA (red spectrum) (shifted 1500 arbitrary units (a. u.) downward); c. Rnase-ethanol/HCl (black spectrum) (shifted 600 a. u. upward). Difference spectra (experimental - fitting curve) after analysis of the amide I Raman bands of lysozyme at different purification steps (Fig. 5, [1]). a. Native lysozyme (blue); b. Lysozyme-TCA (red); c. Lysozyme-ethanol/HCl (black). Difference spectra (experimental – fitting curve) after analysis of the amide I Raman bands of HSA at different purification steps (Fig. 6, [1]). a. Native HSA (blue); b. HSA-TCA (red); c. HSA-ethanol/HCl (black). Relative integrated intensities of lysozyme amide I contribution from peak #6 assigned to unordered structures (uo), peak#7 (ordered α helices, ho), peak#8 (unordered α helices and β sheets, hu+sh), and peak #9 (turns, tu) as obtained after profile fitting of amide I region of the Raman spectra (Fig. 5, Ref. [1]). Values on top of each bar correspond to the Raman shift on which the contribution peak was centred at the end of the fitting. Relative integrated intensities of HSA amide I contribution from peak #1 assigned to unordered structures (uo), peak#2 (ordered α helices, ho), peak#3 (unordered α helices and β sheets, hu+sh), and peak #4 (turns, tu) as obtained after profile fitting of amide I region of the Raman spectra shown in Fig. 6[1]. Values on top of each bar correspond to the Raman shift on which the contribution peak was centred at the end of the fitting. The unfolding or aggregation of proteins usually involves some dynamic changes in their secondary structures. These changes are mainly monitored by the analysis of the amide I region (1600–1690 cm−1) which is assumed to be sensitive to α-helical secondary structures [8].

Extraction and purification of proteins from complex samples: LC-HRMS analysis

We present processed data of UPLC- HRMS analysis of proteins from different samples (mouse liver, river biofilm, soil) after TCA precipitation and solvent purification. The datasets in XML format can be used to evaluate ethanol/HCl purification for proteins profiling. Table 1 gives the HRMS features of peptides targeted for the standard proteins after in silico tryptic digestion. Table 2 presents endogenous proteins identified in soil, biofilm and mouse liver samples after purification following either the designed approach or published protocols (Mascot identification). Table 3 presents endogenous proteins detected in the mouse liver sample and quantified through Skyline with corresponding peptides and transitions for PRM. Table 4 presents endogenous proteins detected in the biofilm sample and quantified through Skyline with corresponding peptides and transitions for PRM (Table 5).

Table 1

HRMS features of peptides targeted for the four standard proteins after in silico tryptic digestion.

Protein name	Peptide sequence	[M+H]¹⁺	[M+2H]²⁺	[M+3H]³⁺	[M+4H]⁴⁺
LYSO-1	FESNFNTQATNR		714.8288	476.8883
LYSO-2	HGLDNYR	874.4166	437.7119	292.1437
RNASE-1	CKPVNTFVHESLADVQAVCS QK			839.7457	630.0611
RNASE-2	HIIVACEGNPYVPVHFDASV		1112.5464	742.0334
RNASE-3	YPNCAYK	915.4029	458.2051
HSA-1	AVMDDFAAFVEK		671.8210	448.2164
HSA-2	LVAASQAALGL	1013.5990	507.3031
HSA-3	YLYEIAR	927.4934	464.2504	309.8360
EXO-1	GSCSTSSGVPAQVESQSPNA K		1039.4764	693.3200
EXO-2	YGTGYCDSQCPR		732.2876	488.5275
EXO-3	VTFSNIK	808.4563	404.7282

Table 2

Endogenous proteins identified in soil, biofilm and mouse liver after purification following either the designed approach or the published protocols.

Sample	Location	Protein name	Phylogenetic origin	Protein coverage (%)		Scorea		GRAVY	MW (Da)b
				The designed approach	Published protocol	The designed approach	Published protocol
Soil	Extracellular region	Endoglucanase EG-II	Hypocrea jecorina	18	19	161	251	−0.19	44883
	Extracellular region	Xyloglucanase	Hypocrea jecorina	1	1	76	114	−0.21	87307

Biofilm	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	C-phycoerythrin alpha chain	Microchaete diplosiphon	29	29	269	239	−0.15	17786
	chloroplast thylakoid membrane ; Peripheral membrane protein By similarity; Stromal side	R-phycoerythrin alpha chain	Porphyra purpurea	20	17	168	119	−0.19	17972
	Cellular thylakoid membrane; Peripheral membrane protein ; Cytoplasmic side	C-phycocyanin-1 alpha chain	Synechococcus sp,	17	17	181	177	−0.11	17335
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	C-phycoerythrin alpha chain	Synechocystis sp,	20	20	209	176	−0.12	17756
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	Allophycocyanin alpha chain 1	Microchaete diplosiphon	11	11	76	84	−0.14	17411
	chloroplast thylakoid membrane ; Peripheral membrane protein ; Stromal side	B-phycoerythrin beta chain	Porphyridium purpureum	21	20	117	183	0.25	18884
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	C-phycoerythrin beta chain	Microchaete diplosiphon	21	16	138	85	0.21	19568
	chloroplast thylakoid membrane ; Peripheral membrane protein ; Stromal side	R-phycoerythrin beta chain	Pyropia haitanensis	23	28	129	144	0.26	18810
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	C-phycocyanin-1 beta chain	Microchaete diplosiphon	16	12	64	122	0.17	18080
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	Allophycocyanin subunit alpha 1	Nostoc sp,	17	19	99	112	−0.09	17392
	chloroplast thylakoid membrane ; Peripheral membrane protein ; Stromal side	C-phycocyanin beta chain	Aglaothamnion neglectum	11	12	112	111	0.09	18290
	NI	Ribulose bisphosphate carboxylase large chain	Trichodesmium erythraeum	5	8	90	122	−0.32	53615
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	Allophycocyanin alpha chain	Anabaena cylindrica	6	11	84	83	0.01	17128
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	C-phycoerythrin alpha chain	Pseudanabaena tenuis	18	18	144	126	−0.24	17780
	chloroplast thylakoid membrane ; Multi-pass membrane protein	Photosystem II CP47 reaction center protein	Odontella sinensis	8	8	117	114	0.08	56436
	NI	Ribulose bisphosphate carboxylase large chain	Cyanothece sp,	9	6	94	89	−0.27	53531
	chloroplast	Ribulose bisphosphate carboxylase large chain (Fragment)	Calyptrosphaera sphaeroidea	5	9	90	107	−0.10	50919
	chloroplast	Ribulose bisphosphate carboxylase large chain	Gracilaria tenuistipitata var, liui	8	10	111	132	−0.10	54442
	chloroplast	Ribulose bisphosphate carboxylase large chain	Cylindrotheca sp,	6	6	109	108	−0.12	54400
	chloroplast thylakoid membrane ; Peripheral membrane protein ; Stromal side	Allophycocyanin beta chain	Cyanidium caldarium	13	16	94	83	−0.04	17574
	chloroplast	Ribulose bisphosphate carboxylase small chain	Antithamnion sp,	5	5	72	72	−0.58	16247
	NI	Carbon dioxide-concentrating mechanism protein CcmK homolog 1	Synechocystis sp,	18	29	71	72	−0.19	11128
	chloroplast thylakoid membrane ; Peripheral membrane protein ; Stromal side	R-phycoerythrin beta chain	Aglaothamnion neglectum	7	7	100	69	0.27	18710
	chloroplast	Ribulose bisphosphate carboxylase large chain (Fragment)	Haptolina hirta	9	10	141	139	−0.11	51098
	chloroplast	Ribulose bisphosphate carboxylase large chain	Antithamnion sp,	7	7	117	113	−0.12	54372
	Cellular thylakoid membrane ; Peripheral membrane protein ; Cytoplasmic side	Allophycocyanin beta chain	Thermosynechococcus elongatus	18	18	103	121	0.10	17462
	Cell inner membrane ; Multi-pass membrane protein	Photosystem I P700 chlorophyll a apoprotein A2	Gloeobacter violaceus	2	2	78	75	0.15	96126
	chloroplast thylakoid membrane; Peripheral membrane protein; Stromal side	Phycobiliprotein ApcE	Aglaothamnion neglectum	1	1	73	72	−0.23	101319
	NI	Ribulose bisphosphate carboxylase large chain	Synechocystis sp,	6	6	120	117	−0.29	53084
	chloroplast thylakoid membrane; Peripheral membrane protein; Stromal side	Allophycocyanin beta chain	Galdieria sulphuraria	16	16	96	73	0.02	17536

Mouse liver	Nucleus, Mitochondrion	Carbamoyl-phosphate synthase	Mus musculus	39	33	1637	1268	−0.12	165711
	Cytoplasm	Arginase-1	Mus musculus	29	35	300	310	−0.19	34957
	Cytosol, Nucleus,Membrane	Selenium-binding protein	Mus musculus	31	28	526	405	−0.31	53147
	Cytoplasm	Argininosuccinate synthase	Mus musculus	32	15	429	191	−0.11	46840
	Mitochondrion	Glyceraldehyde-3-phosphate dehydrogenase	Mus musculus	31	32	321	298	−0.04	36072
	cytosol	Cytosolic 10-formyltetrahydrofolate dehydrogenase	Mus musculus	9	17	139	361	−0.36	99502
	Extracellular region	3-ketoacyl-CoA thiolase, mitochondrial	Mus musculus	10	20	137	216	−0.38	42260
	Nucleus, Cytoskeleton,Cytosol	Serum albumin	Mus musculus	15	18	327	349	−0.09	70700
	Cytoplasm	Alcohol dehydrogenase 1	Mus musculus	19	29	161	212	0.20	40601
	membrane	Aspartate aminotransferase, mitochondrial	Mus musculus	15	16	231	215	−0.23	47780
	Endoplasmic reticulum	Carboxylesterase 3B	Mus musculus	12	14	201	183	−0.12	63712
	Cytoplasm	Glycine N-methyltransferase	Mus musculus	29	19	131	127	−0.25	33110
	membrane	Cytochrome P450 2D10	Mus musculus	9	2	100	123	−0.06	57539
	Cytoplasm	Aspartate aminotransferase, cytoplasmic	Mus musculus	7	13	112	115	−0.25	46504
	Cytoplasm	Adenosylhomocysteinase	Mus musculus	27	14	335	120	−0.07	47780
	Cytosol	Fructose-1,6-bisphosphatase 1	Mus musculus	12	16	117	120	−0.12	37288
	Endoplasmic reticulum	Carboxylesterase 3A	Mus musculus	13	9	220	139	−0.12	63677
	Mitochondrion	Sarcosine dehydrogenase, mitochondrial	Mus musculus	8	6	182	209	−0.25	102644
	membrane	UDP-glucuronosyltransferase 1-1	Mus musculus	4	8	94	141	0.09	60749
	Cytosol	Hemoglobin subunit beta-1	Mus musculus	16	24	111	105	0.08	15944
	Peroxisome	Peroxisomal bifunctional enzyme	Mus musculus	3	2	98	78	−0.12	78822
	membrane	Microsomal glutathione S-transferase	Mus musculus	17	21	80	87	0.15	17597
	membrane	Cytochrome P450 2F2	Mus musculus	6	7	128	130	−0.13	56141
	NI	Pyrethroid hydrolase Ces2a	Mus musculus	9	5	100	76	_	57539
	Extracellular region	Homogentisate 1,2-dioxygenase	Mus musculus	6	6	81	114	−0.34	50726
	Cytoplasm	Regucalcin	Mus musculus	4	13	72	112	−0.28	33899
	Peroxisome	3-ketoacyl-CoA thiolase B, peroxisomal	Mus musculus	13	8	116	84	0.05	44481
	membrane	Sorbitol dehydrogenase	Mus musculus	6	6	90	89	0.06	38795
	membrane	ATP synthase subunit f, mitochondrial	Mus musculus	26	26	70	71	−0.30	10394
	membrane	ATP synthase subunit alpha, mitochondrial	Mus musculus	14	10	193	160	−0.10	59830
	Cytosol	Urocanate hydratase	Mus musculus	2	1	100	76	−0.14	75227
	Extracellular region	Fumarylacetoacetase	Mus musculus	3	6	75	74	−0.21	46488
	Mitochondrion; Peroxisome	Uricase	Mus musculus	17	11	157	97	−0.46	35245
	Cytoskeleton	Fructose-bisphosphate aldolase B	Mus musculus	15	13	180	119	−0.26	39938
	membrane	UDP-glucuronosyltransferase 2B17	Mus musculus	11	6	104	96	−0.03	61386
	NI	Pyrethroid hydrolase	Mus musculus	9	7	108	89	−0.08	62356
	Cytoplasm	3-hydroxyanthranilate 3,4-dioxygenase	Mus musculus	9	6	90	87	−0.55	32955
	Mitochondrion	Hydroxymethylglutaryl-CoA synthase, mitochondrial	Mus musculus	7	6	86	70	−0.34	57300
	Mitochondrion	Trifunctional enzyme subunit alpha, mitochondrial	Mus musculus	9	7	90	81	−0.10	83302
	Endoplasmic reticulum	Microsomal triglyceride transfer protein large subunit	Mus musculus	1	1	74	80	−0.16	99664
	membrane	Cytochrome b-c1 complex subunit 2, mitochondrial	Mus musculus	4	4	73	76	−0.06	48262

MASCOT score greater than 67.

MW: Molecular weight.

Table 3

Endogenous peptides and transitions for PRM methods.

			PRM
Protein name	Abreviattion	Peptide	Precursor(m/z)	Product (m/z)
Carbamoyl-phosphate synthase	CPSM	TAVDSGIALLTNFQVTK	898.4844	950.5306
				837.4465
				736.3988
		VLGTSVESIMATEDR	804.4009	1051.4725
				722.3138
				591.2733
		AFAMTNQILVER	696.8688	972.5473
				516.3140
				403.2300
		GQNQPVLNITNR	677.3653	926.5418
				617.3365
				390.2096
		AADTIGYPVMIR	653.8448	835.4495
				615.3647
				472.2402
		EPLFGISTGNIITGLAAGAK	644.0263	801.4829
				688.3988
				587.3511
		IALGIPLPEIK	582.3735	696.4291
				355.2340
				468.3180
		VMIGESIDEK	560.7814	890.4466
				777.3625
				231.1162
		SVGEVMAIGR	509.7711	832.4345
				646.3705
				547.3021

Argininosuccinate synthase	ASSY	EQGYDVIAYLANIGQK	891.4571	977.5415
				743.4410
				630.3570
		FELTCYSLAPQIK	785.4027	1085.4972
				556.3453
				485.3082
		QHGIPIPVTPK	593.8508	921.5768
				751.4713
				541.3344
		NQAPPGLYTK	544.7904	846.472
				775.4349
				314.1459
		YLLGTSLARPCIAR	530.9643	657.8692
				601.3271
				277.1547

Selenium-binding protein 2	SBP2	GSFVLLDGETFEVK	770.8983	1037.515
				924.4309
				809.404
		EEIVYLPCIYR	727.871	984.4971
				821.4338
				708.3498
		LTGQIFLGGSIVR	680.901	848.4989
				701.4304
				588.3464
		IYVVDVGSEPR	617.3273	957.5
				858.4316
				545.2678
		IFVWDWQR	575.2956	889.4315
				790.3631
				261.1598
		VIEASEIQAK	544.3033	875.4469
				746.4043
				675.3672

Glyceraldehyde-3-phosphate dehydrogenase	G3P	VPTPNVSVVDLTCR	778.9087	1259.6412
				949.4771
				630.3243
		WGEAGAEYVVESTGVFTTMEK	764.3561	912.4495
				892.4123
				756.3597
		GAAQNIIPASTGAAK	685.3753	815.4621
				702.3781
				668.3726
		LISWYDNEYGYSNR	593.9373	1021.4625
				539.2572
				376.1939

Arginase-1	ARGI1	VMEETFSYLLGR	722.8607	1214.6052
				855.4723
				708.4039
		EGLYITEEIYK	679.3479	1058.5405
				895.4771
				782.3931
		VSVVLGGDHSLAVGSISGHAR	673.3641	866.9581
				817.4239
				760.8819
		SLEIIGAPFSK	581.3293	606.3246
				556.3341
				478.266

Table 4

Endogenous peptides and transitions for PRM methods.

			PRM
Protein name	Abreviattion	Peptide	Precursor(m/z)	Product (m/z)
R-phycoerythrin alpha chain, Porphyra purpurea	PHEA_PORPU	SVITTTISAADAAGR	717.3834	1134.5749
				1033.5273
				374.2146
		FPSSSDLESVQGNIQR	588.6235	715.3846
				621.2515
				587.3260
		NPGEAGDSQEK	566.2493	920.3956
				663.2944
				491.2460

C-phycocyanin-1 alpha chain, Synechococcus sp.	PHCA1_SYNP6	TPLTEAVAAADSQGR	743.8784	1175.5651
				945.4748
				775.3693
		FLSSTELQVAFGR	727.8855	1194.6113
				1107.5793
				790.457

C-phycoerythrin alpha chain, Synechocystis sp.	PHEA_SYNY1	TLGLPTAPYVEALSFAR	602.6647	1152.6048
				793.4203
				664.3777
		FPSTSDLESVQGSIQR	584.2917	688.3737
				635.2671
				560.3151

C-phycoerythrin alpha chain, Microchaete diplosiphon	PHEA_MICDP	SVVTTVIAAADAAGR	701.3834	1116.6008
				815.437
				374.2146
		ALGLPTAPYVEALSFAR	592.6612	1152.6048
				793.4203
				664.3777
		FPSTSDLESVQGSIQR	584.2917	688.3737
				635.2671
				560.3151

Table 5

Total spectrum, peptide and protein counts after purification by our approach versus published protocols on complex matrices.

	Total spectrum count	Peptide count	Protein count
Biofilm-published approacha	932	585	195
Biofilm-our approacha	937	424	163
Mouse liver-published approacha	1122	1408	416
Mouse liver-our approacha	959	1205	355
Soil-published approachb	946	293	72
Soil-our approachb	932	488	128

Data from the ProteomeXchange Consortium via the PRIDE [10] repository with the dataset identifier PXD0081110 and 10.6019/PXD008110.

Average of three replicates.

Counts of a single replicate.

HRMS features of peptides targeted for the four standard proteins after in silico tryptic digestion. Endogenous proteins identified in soil, biofilm and mouse liver after purification following either the designed approach or the published protocols. MASCOT score greater than 67. MW: Molecular weight. Endogenous peptides and transitions for PRM methods. Endogenous peptides and transitions for PRM methods. Total spectrum, peptide and protein counts after purification by our approach versus published protocols on complex matrices. Data from the ProteomeXchange Consortium via the PRIDE [10] repository with the dataset identifier PXD0081110 and 10.6019/PXD008110. Average of three replicates. Counts of a single replicate.

Experimental design, materials and methods

Experimental design and materials and methods have been reported previously [1].

Subject area	Chemistry
More specific subject area	Proteomics, protein purification, protein precipitation, trichloroacetic acid
Type of data	Tables, Figures
How data was acquired	Raman (LabRAM HR800UV confocal microspectrometer, Horiba Jobin Yvon, Kyoto, Japan)
	Bradford assay (DC Protein Assay, Biorad)
	Electrophoresis (ImageJ software)
	UPLC-HRMS (Accela LC pumps, Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer equipped of an ESI source, Thermo Fisher Scientific, Waltham, MA, USA)
	MASCOT search engine (Matrix Science, London, UK; version 2.6.0) and Skyline software (MacCoss Lab, Washington, US; version 3.7.0.10940)
	ProteomeXchange Consortium with identifier PXD008110
Data format	Raw, analyzed and processed data
Experimental factors
Experimental features	Proteins extraction was performed on 500 mg of soil, 10 mg of biofilm and 15 mg of mouse liver as starting material according to protocols of Chourey et al.[2], Huang et al.[3]and Song et al.[4]respectively.
	Proteins were precipitated with 25% (w/v) trichloroacetic acid (TCA).
	The washing of protein pellet was performed with three different agents (acetone, ethanol, or ethanol/HCl). The mixture was vortexed and kept at −20 °C for 1 h, centrifuged at 16,600 g for 15 min at 4 °C. The resulting pellets were dried in a SpeedVac concentrator, solubilized in a 50 mM of ammonium bicarbonate buffer containing 10 mM of Tris. Proteins were subjected to trypsin digestion for 24 h at 37 °C. Digestion was stopped with formic acid before gel, bradford and mass analysis.
Data source location	Poitiers, France
Data accessibility	data are with this article

8 in total

1. Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis.

Authors: D I Jacobs; M S van Rijssen; R van der Heijden; R Verpoorte
Journal: Proteomics Date: 2001-11 Impact factor: 3.984

2. Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.

Authors: Ewelina Fic; Sylwia Kedracka-Krok; Urszula Jankowska; Artur Pirog; Marta Dziedzicka-Wasylewska
Journal: Electrophoresis Date: 2010-10 Impact factor: 3.535

3. Direct cellular lysis/protein extraction protocol for soil metaproteomics.

Authors: Karuna Chourey; Janet Jansson; Nathan VerBerkmoes; Manesh Shah; Krystle L Chavarria; Lauren M Tom; Eoin L Brodie; Robert L Hettich
Journal: J Proteome Res Date: 2010-11-15 Impact factor: 4.466

4. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis.

Authors: Lei Jiang; Lin He; Michael Fountoulakis
Journal: J Chromatogr A Date: 2004-01-16 Impact factor: 4.759

5. Comparative Proteomics Provides Insights into Metabolic Responses in Rat Liver to Isolated Soy and Meat Proteins.

Authors: Shangxin Song; Guido J Hooiveld; Wei Zhang; Mengjie Li; Fan Zhao; Jing Zhu; Xinglian Xu; Michael Muller; Chunbao Li; Guanghong Zhou
Journal: J Proteome Res Date: 2016-02-25 Impact factor: 4.466

6. Study of a novel agent for TCA precipitated proteins washing - comprehensive insights into the role of ethanol/HCl on molten globule state by multi-spectroscopic analyses.

Authors: Balkis Eddhif; Justin Lange; Nadia Guignard; Yann Batonneau; Jonathan Clarhaut; Sébastien Papot; Claude Geffroy-Rodier; Pauline Poinot
Journal: J Proteomics Date: 2017-11-27 Impact factor: 4.044

7. Total protein extraction for metaproteomics analysis of methane producing biofilm: the effects of detergents.

Authors: Hung-Jen Huang; Wei-Yu Chen; Jer-Horng Wu
Journal: Int J Mol Sci Date: 2014-06-06 Impact factor: 5.923

8. 2016 update of the PRIDE database and its related tools.

Authors: Juan Antonio Vizcaíno; Attila Csordas; Noemi del-Toro; José A Dianes; Johannes Griss; Ilias Lavidas; Gerhard Mayer; Yasset Perez-Riverol; Florian Reisinger; Tobias Ternent; Qing-Wei Xu; Rui Wang; Henning Hermjakob
Journal: Nucleic Acids Res Date: 2015-11-02 Impact factor: 16.971

8 in total

2 in total

Review 1. Soil Metaproteomics for the Study of the Relationships Between Microorganisms and Plants: A Review of Extraction Protocols and Ecological Insights.

Authors: Maria Tartaglia; Felipe Bastida; Rosaria Sciarrillo; Carmine Guarino
Journal: Int J Mol Sci Date: 2020-11-11 Impact factor: 5.923

2. Magnetic nanoparticles coated with carboxylate-terminated carbosilane dendrons as a reusable and green approach to extract/purify proteins.

Authors: Isabel M Prados; Andrea Barrios-Gumiel; Francisco J de la Mata; M Luisa Marina; M Concepción García
Journal: Anal Bioanal Chem Date: 2021-12-09 Impact factor: 4.142

2 in total