The first global screening of protein substrates bearing protein-bound 3,4-dihydroxyphenylalanine in E. coli and human mitochondria

Abstract

Protein hydroxylation at proline and lysine residues is known to have important effects on cellular functions, such as the response to hypoxia. However, for protein hydroxylation at tyrosine residues (called protein-bound 3,4-dihydroxy-phenylalanine (PB-DOPA) has not been carefully examined. Here we report the first proteomics screening of the PB-DOPA protein substrates and their sites in E. coli and human mitochondria by nano-LC/MS/MS and protein sequence alignment using the PTMap algorithm. Our study identified 67 novel PB-DOPA sites in 43 E. coli proteins, and 9 novel PB-DOPA sites in 7 proteins from HeLa mitochondria. Bioinformatics analysis indicates that the structured region is more favored than the unstructured regions of proteins for the PB-DOPA modification. The PB-DOPA substrates in E. coli were dominantly enriched in proteins associated with carbohydrate metabolism. Our study showed that PB-DOPA may be involved in regulation of the specific activity of certain evolutionarily conserved proteins such as superoxide dismutase and glyceraldehyde 3-phosphate dehydrogenase, suggesting the conserved nature of the modification among distant biological species. The substrate proteins identified in this study offer a rich source for hunting their regulatory enzymes, and for further characterization of the possible contributions of this modification to cellular physiology and human diseases.

INTRODUCTION

Protein hydroxylation has been found in multiple amino acid residues, including proline, lysine, aspartate, asparagine, tryptophan, and tyrosine ¹. Members of the Fe(II)- and 2-oxoglutarate-dependent family of dioxygenases can catalyze these hydroxylation reactions ². Emerging evidence suggest that this family of post-translational modifications (PTMs) play structural roles, and have important cellular functions. For example, prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor HIFα, the master regulator of the cellular hypoxic response pathway ^{3, 4}. This protein is associated with diverse physiological and pathophysiological conditions, such as angiogenesis, myocardial ischemia, and neuronal ischemia ⁵. U2AF65 (U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit) can be lysine-5-hydroxylated by the Fe(II) and Jmid6 (2-oxoglutarate-dependent dioxygenase Jumonji domain-6 protein); and this modification has a selective effect on the RNA splicing ⁶. Hydroxylation of proline in the newly synthesized collagen polypeptide chains is essential for the formation of the triple-helical molecules ⁷. Given the critical roles of the hydroxylation reactions at proline, lysine, and asparagine residues in regulating proteins’ and cellular functions, it is anticipated that hydroxylation at the tyrosine residue will also play a role in cellular physiology.

The tyrosine residue can be oxidized to the protein-bound 3,4-dihydroxyphenylalanine residue (PB-DOPA, or 3-hydroxytyrosine) by enzyme-catalyzed reaction. PB-DOPA can be generated by oxidation of tyrosine residue by tyrosinase or tyrosine oxidase, which are present in neuronal cells and melanocytes of skin, hair follicles, and pigmented epithelial cells of the retina ⁸. PB-DOPA can be also produced by the enzymatic reactions of bacterial tyrosinase as cresolase activity, and by autooxidation in Streptomyces species ^{9, 10}. Moreover, PB-DOPA could be generated from L-DOPA ^{11, 12} which is a precursor to the biological pigment melanin and is also the precursor to the neurotransmitters dopamine, norepinephrine, and epinephrine ^{8, 13, 14}. In addition, the L-DOPA has been used in clinics for treating Parkinson’s diseases and dopamine-responsive dystonia, in order to increase dopamine concentration in the brains of patients ¹⁵. Limited studies suggest that PB-DOPA is associated with cellular functions.

Determining the substrates of PTMs, and their modification sites, are typically the first steps in study of PTM biology. The history of investigations into protein lysine acetylation provides a good example of substrate identification and functional characterization of a PTM, which the study of protein tyrosine hydroxylation is likely to follow. Lysine acetylation (K^Ac) was initially identified in histones in 1964 ¹⁶. The first non-histone substrate protein, p53, was identified in 1997 ¹⁷. Initially, K^Ac was thought to be restricted to nuclei because of its initial identification and characterization within histones and transcription factors. However, this paradigm was challenged after the discovery of the first K^Ac cytosolic proteins ^{18, 19}. Identification of diverse substrates in both cytosolic and mitochondrial fractions ²⁰, and demonstration of the class III histone deacetylases in mitochondria ¹⁹, conclusively established the substrate and functional diversities of this PTM. A proteomics screen in mitochondria gave the surprising result that more than 20% of mitochondrial proteins are lysine acetylated in mammalian cells ²⁰, indicating the role of this modification in mitochondria and metabolism ²⁰.

Here, we presented the first systematic screening of PB-DOPA substrates and sites in E. coli and HeLa mitochondria by protein separation, nano-LC/MS/MS, and the PTMap for sequence alignment. Our studies identified 67 novel sites of PB-DOPA in 43 E. coli proteins and 9 novel sites of PB-DOPA in 7 HeLa mitochondrial proteins, present in 2.5% and 0.5% of proteins in E. coli and HeLa mitochondria, respectively. We verified the identification of PB-DOPA by the purified recombinant proteins and MS/MS of the corresponding synthetic peptides. Spectral counting comparison between the modified peptides and their corresponding unmodified counterparts showed that the PTM stoichiometry can be as high as 32.7%. In addition, the E. coli proteins bearing PB-DOPA are highly enriched in cellular metabolism, especially carbohydrate metabolism. This study therefore revealed a previously unexpected role of PB-DOPA in prokaryotic biochemical pathways, and provided evidence that PB-DOPA is an abundant and evolutionarily conserved modification.

EXPERIMENTAL PROCEDURE

Materials

Modified porcine trypsin was purchased from Promega Inc. (Madison, WI). LB-medium was purchased from MP Biomedicals LLC (Solon, Ohio) and Ni-NTA Agarose was a product of QIAGEN GmBH (Valencia, CA). Trichloroacetic acid (TCA) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO). LC/MS grade water and acetonitrile (ACN) were purchased from Honeywell Burdick & Jackson (Muekegon, MI). All other chemicals were of the highest purity available or analytical grade. All the peptides used in this study were synthesized by Genemed Synthesis Inc. (San Antonio, TX).

Preparation of E. coli lysate and mitochondrial fractions from HeLa S3 cell

E. coli K-12 DH10B (Invitrogen, Carlsbad, CA) was cultured in LB media to OD600 = 0.7. The cells were harvested and washed twice with cold PBS. The cell pellet was lysed in NETN buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 0.5% NP40, 20 mM beta-mercaptoethanol, pH 8.0, with the protease cocktail inhibitors) by sonication. The lysate was dialysed against 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.0 for 1.5 hrs at 4 °C and then centrifuged at 20,000 x g for 20 minutes. Supernatant was collected for HPLC separation.

Mitochondria were isolated from HeLa S3 cells (Biovest International Inc. Minneapolis, MN) using a discontinuous Percoll gradient as previously reported ²¹. The mitochondria were lysed by HIM buffer (200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES, pH 7.5, with the protease cocktail inhibitors) containing 1% n-dodecyl-D-maltoside on ice with frequent vortexing for 30 mins. Samples were centrifuged at 50,000 x g for 30 mins. The supernatant was collected for HPLC fractionation.

Fractionation of protein lysates

A preparative HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) was used for protein fractionation. 10-20 mg of a protein lysate of interest was loaded onto a PolyCATWAX column (200 × 9.4mm, 5 m, 1000Å, PolyLC Inc., Columbia, MD). The proteins were eluted using a HPLC gradient from 0% buffer B to 100% buffer B in buffer A for 56 min (mobile buffer A: 10 mM MES, pH 6.0; and mobile buffer B: 10 mM MES, 800 mM NaCl, pH 6.0) at a flow rate of 4 ml/min at 4 °C. HPLC elution was collected every 2 min. The proteins in each fraction were precipitated by conventional trichloroacetic acid (TCA)/acetone method and air dried.

Nano-HPLC/mass spectrometric analysis

The tryptic digest from each fraction was analyzed in an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The peptides were separated in a home-made capillary HPLC column (100 mm length × 75 μm internal diameter, 5 μm particle size, 100 Å pore diameter) with Jupiter C₁₂ resin (Phenomenex, St. Torrance, CA) using a gradient from 2% to 30% solvent B in solvent A (mobile phase A: 0% ACN in 0.1% formic acid; and mobile phase B: 100% ACN in 0.1% formic acid) for 100 min. The eluted peptides were directly electrosprayed into the mass spectrometer using a nanospray source. The MS was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS (MS²) acquisition. Survey full scan MS spectra (from m/z 350-1800) were acquired in the Orbitrap with resolution R=30,000 at m/z=400. The 10 most intense ions were sequentially isolated for fragmentation in the linear ion trap using collsionally induced dissociation. The following parameters were specified: dynamic exclusion: 36 seconds; the repeat count: 2, and the exclusion window: +2 and −1 Da.

Protein sequencing alignment

All MS/MS spectra were searched against the Refseq protein sequence database for E. coli sub str. K12DH10B (4127 sequences) using Mascot and PTMap software ²². The specific parameters for protein sequence database searching included tyrosine hydroxylation, methionine oxidation, and cysteine alkylation as a variable modification, trypsin as the digesting enzyme, three allowed missing cleavages, and mass error of 15 ppm for precursor ions and 0.6 Da for fragment ions. Charge states of +2 and +3 were considered for parent ions. The ions of the single charge state was not considered, because single charged ions represent lower quality data than ions with +2 and +3 charges. If more than one spectrum was assigned to a peptide, only the spectrum with the highest PTMap score was selected for manual analysis. False discovery rate was estimated using reverse-decoy database strategy by appending a reversed database to the forward database ²³. All peptides bearing PB-DOPA identified with peptide score of PTMap > 1.0 (FDR < 5%) were manually examined with the rules described previously ²⁴.

PTM stoichiometry (%) used in this study is the ratio of the number of MS/MS spectra for the peptide bearing the PB-DOPA residue divided by the number of MS/MS spectra for the both peptides bearing the corresponding Tyr site, namely the unmodified peptides with or without missing cleavages (cutoff PTMap score > 0.5, FDR < 1%) and modified peptides (cutoff PTMap score > 1.0, FDR < 5%).

Bioinformatic analysis

Secondary structures of E.coli proteins were predicted using GOR algorithm ²⁵. Biological processes enrichment analysis was performed using Gostats package in the statistical environment R. Probability values (p) of all enriched processes (p<0.01) were first converted into L values: L = -Log₁₀(p) and then Z scores were calculated using the following formula: Z = [L-Ave(L)] / Std(L), where Ave(L) and Std(L) refer to the average and the standard deviation of all L values. Clustering of the biological processes was performed by one-way hierarchical clustering function in Genesis software ²⁶. Evolution conservation analysis was performed using blast search of each PB-DOPA protein against the RefSeq protein database of the six model species – yeast (taxonomy ID: 4932), C. elegans (taxonomy ID: 6239), Drosophila (taxonomy ID: 7227), zebra fish (taxonomy ID: 7955), mouse (taxonomy ID: 10090), human (taxonomy ID: 9606).

MS/MS of synthetic peptides bearing a PB-DOPA residue

The peptides bearing a PB-DOPA residue were synthesized by Genemed Synthesis Inc (San Antonio, TX). All synthetic peptides were verified by MS and MS/MS using the LTQ-XL (Thermo Fisher Scientific, Waltham, MA) coupled with a nano-HPLC system (Agilent 1200 series Nanoflow, Agilent Technologies, Santa Clara, CA). During HPLC/MS/MS analysis, loading of a synthetic peptide was adjusted close to that of its corresponding in vivo peptides. Extensive washing was carried out to avoid peptide carry-over. To verify coelution of the in vivo peptide and its synthetic counterpart, the in vivo peptide bearing a PB-DOPA residue and its corresponding synthetic peptide were monitored by extracted ion chromatogram of specific product ion. The nano-HPLC/mass spectrometric analysis was carried out using the procedure described in Materials and Methods.

Expression and purification of the recombinant proteins

The bacteria strain of interest containing a plasmid expressing an His₆-tagged protein of interest, originally developed by National BioResource Project in Japan ²⁷, was grown in LB media with chloramphenicol (30 μg/ml). The cells were treated with isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.04 mM) to stimulate the protein expression for 3 hr, and harvested by centrifugation at 5,000 x g at 4 °C for 20 min. The pellet was washed with 5 ml of cold PBS, resuspended in a lysis buffer (10 mM imidazole with proteinase inhibitor cocktail) and sonicated four times at 4 °C for 10 s each time. Unbroken cells and cellular debris were removed by centrifugation at 20,000 x g at 4 °C for 20 min. Finally, the His₆-tagged protein was purified under non-denaturing conditions by a standard procedure using Ni-NTA agarose (Qiagen, Valencia, CA). The purified proteins were resolved in SDS PAGE. The protein band of interest was excised, and in-gel digested for nano-HPLC/mass spectrometric analysis.

In-gel digestion of purified proteins with trypsin

The purified His₆-tagged proteins were separated by SDS-PAGE and visualized by staining with colloidal Coomassie blue. The protein bands of interest were excised, and in-gel digested using the clean protocol previously described ²⁸.

RESULTS

Strategy

To identify protein-bearing PB-DOPA residues, the E. coli and HeLa mitochondrial protein lysate were separated into 55 fractions in a mixed-bed PolyCATWAX column (Figure 1A). The proteins in each fraction were precipitated using the TCA/acetone method, and tryptically digested ²⁹. The resulting proteolytic peptides were analyzed by nano-HPLC/LTQ Orbitrap mass spectrometer. The MS/MS data were first analyzed by Mascot algorithm to identify proteins. The MS/MS data and the identified proteins were then used as inputs, and analyzed by PTMap algorithm to identify PB-DOPA peptides and sites (Figures 1B and 1C). The candidate peptides bearing the PB-DOPA residue were further examined manually as previously described, to ensure the exclusive localization of oxidation sites in tyrosine instead of in other adjacent amino acid residues ^{24, 30}.

Figure 1.

Strategy. Procedure for global screening of PB-DOPA in E. coli and HeLa mitochondria (A), MS (B) and MS/MS spectra of an PB-DOPA-containing peptide (LEQY^OHFDR) from ribosomal hibernation promoting factor in E. coli (C).

The analysis identified 1,733 and 1,355 proteins from E. coli and HeLa mitochondrial protein lysates, respectively. Among these proteins, we identified 67 PB-DOPA sites in 43 E. coli proteins, and 9 sites in 7 HeLa mitochondrial proteins (Tables 1 and 2, Supplementary_table 1 and Supplementary_figures 1 and 2). These results suggest that about 2.5% and 0.5% proteins were tyrosine hydroxylated in E. coli and HeLa mitochondria, respectively. Given the fact that a peptide bearing a PB-DOPA typically has lower abundance, and will be more difficult to detect in HPLC/MS/MS analysis than its corresponding unmodified counterpart, the real abundance of PB-DOPA should be significantly higher than the percentage calculated above.

Table 1.

List of peptides bearing PB-DOPA in E. coli lysate

No	Sequencesa	Stochiometryb		GI number	Protein Information
		Scan #	%
1	KLSYTGEVK*	2/8	20.0%	170079671	transaldolase_B
2	LSYDTEASIAK*	1/7	12.5%	170079671	transaldolase_B
3	YLEHR	1/31	3.1%	170079751	pyruvate_dehydrogenase,_decarboxylase_component_E1
4	EQVAYYKEDEK*	6/37	13.9%	170079751	pyruvate_dehydrogenase,_decarboxylase_component_E1
5	YAMIGDPTGALTR	1/39	2.5%	170080288	alkyl_hydroperoxide_reductase,_C22_subunit
6	HYGALQGLNK*	5/29	14.7%	170080415	phosphoglyceromutase_1
7	AIDFSDGYYK*	4/17	19.0%	170080470	glutamine_ABC_transporter_periplasmic_binding_protein
8	SGTGSVDYAK*	2/7	22.2%	170080470	glutamine_ABC_transporter_periplasmic_binding_protein
9	ADAVLHDTPNILYFIK*	3/29	9.4%	170080470	glutamine_ABC_transporter_periplasmic_binding_protein
10	QFPNIDNAYMELGTNR*	3/23	11.5%	170080470	glutamine_ABC_transporter_periplasmic_binding_protein
11	ATNLLYTR*	14/100	12.3%	170080471	Fe-binding_and_storage_protein
12	YAIVANDVR	6/87	6.5%	170080471	Fe-binding_and_storage_protein
13	YTSVDQLK*	2/9	18.2%	170080522	arginine_ABC_transporter_periplasmic-binding_protein
14	IEYVYQSAEQLR	3/40	7.0%	170080661	glucose-1-phosphatase/inositol_phosphatase
15	YQQEPGVSGPLK	1/44	2.2%	170080661	glucose-1-phosphatase/inositol_phosphatase
16	NVAKPLVSYIDK*	1/10	9.0%	170080661	glucose-1-phosphatase/inositol_phosphatase
17	TVTYDFER	1/21	4.5%	170080787	isocitrate_dehydrogenase,_specific_for_NADP+
18	YYQGTPSPVK	1/46	2.1%	170080787	isocitrate_dehydrogenase,_specific_for_NADP+
19	LKDGEDPGYTLYDLSER	1/87	1.1%	170081164	glutamate_decarboxylase_B,_PLP-dependent
20	YLSDHPK	1/63	1.6%	170081164	glutamate_decarboxylase_B,_PLP-dependent
21	TPEGYASGSLGPTTAGR	1/22	4.3%	170081276	fumarate_hydratase,_aerobic_class_I
22	DALAPHISAETIEYHYGK	1/33	2.9%	170081319	superoxide_dismutase,_Fe
23	HHQTYVTNLNNLIK*	6/21	22.2%	170081319	superoxide_dismutase,_Fe
24	EITSTDDFYR*	2/2	100%	170081336	pyruvate_kinase_I
25	LNFSHGDYAEHGQR	1/7	12.5%	170081336	pyruvate_kinase_I
26	AATYEQIK*	2/5	28.6%	170081435	glyceraldehyde-3-phosphate_dehydrogenase_A
27	LVSWYDNETGYSNK	1/35	2.8%	170081435	glyceraldehyde-3-phosphate_dehydrogenase_A
28	VSQASDSYYYR	2/55	3.5%	170081885	hypothetical_protein_ECDH10B_2427
29	HLVDLYQQQGVEK*	4/22	15.4%	170082073	transaldolase_A
30	TYQQQVAK	2/38	5.0%	170082161	serine_hydroxymethyltransferase
31	AGYAEDEVVAVSK	1/41	2.4%	170082188	pyruvate_formate_lyase_subunit
32	LGDIEYR	2/34	5.6%	170082188	pyruvate_formate_lyase_subunit
33	YPQLTIR	1/116	0.9%	170082188	pyruvate_formate_lyase_subunit
34	DAGYTAVISHR	2/124	1.6%	170082351	enolase
35	QLGVSYFLER	2/51	3.8%	170082411	5-keto_4-deoxyuronate_isomerase
36	HYHESR*	2/16	11.1%	170082464	glycine_decarboxylase,_PLP-dependent,_subunit
37	ANEAYLQGQLGNPK	1/35	2.8%	170082482	fructose-bisphosphate_aldolase,_class_II
38	DSVSYGVVK	1/19	5.0%	170082482	fructose-bisphosphate_aldolase,_class_II
39	YYDPR*	3/15	16.7%	170082482	fructose-bisphosphate_aldolase,_class_II
40	DSQEYVSK	2/28	6.7%	170082482	fructose-bisphosphate_aldolase,_class_II
41	SLYEADLVDEAKR*	4/35	10.3%	170082483	phosphoglycerate_kinase
42	EVHIEGYTPEDKK*	1/10	9.1%	170082514	YggX
43	IPLLIHQPSYNLLNR*	3/24	8.1%	170082547	aldo-keto_reductase
44	IYEAAR*	1/9	10.0%	170082555	alcohol_dehydrogenase,_NAD(P)-dependent_
45	DLADKYGK*	1/5	20.0%	170082556	2,5-diketo-D-gluconate_reductase_A
46	AYSEAVK	1/15	6.3%	170082572	quinol_monooxygenase
47	YTQLIER	2/22	8.3%	170082702	30S_ribosomal_subunit_protein_S15
48	LEQYFDR*	2/7	22.2%	170082737	ribosomal_hibernation_promoting_factor
49	YADEVVR	2/25	7.4%	170082825	50S_ribosomal_subunit_protein_L6_
50	KNPQKNLYTFK	3/96	3.0%	170083020	acid-resistance_protein
51	LVSSAGTGHFYTTTK	1/32	3.0%	170083144	50S_ribosomal_subunit_protein_L33
52	NIFGYQYTIPTHQGR	8/153	5.0%	170083210	tryptophanase/L-cysteine_desulfhydrase
53	YADMLAMSAK	2/100	2.0%	170083210	tryptophanase/L-cysteine_desulfhydrase
54	AYREEAIIK	1/124	0.8%	170083210	tryptophanase/L-cysteine_desulfhydrase
55	FAENAYFIK	6/135	4.3%	170083210	tryptophanase/L-cysteine_desulfhydrase
56	GDEAYSGSR	1/67	1.5%	170083210	tryptophanase/L-cysteine_desulfhydrase
57	SYYALAESVK*	9/75	10.7%	170083210	tryptophanase/L-cysteine_desulfhydrase
58	VLYGALPR*	2/11	15.4%	170083249	sugar_ABC_transporter_ATP-binding_protein
59	IAGDYIAK*	16/91	15.0%	170083251	sugar_ABC_transporter_periplasmic-binding_protein
60	YPVDLK*	17/35	32.7%	170083251	sugar_ABC_transporter_periplasmic-binding_protein
61	RPDYIK	1/26	3.7%	170083379	superoxide_dismutase,_Mn
62	EAGVYMR	1/42	2.3%	170083445	50S_ribosomal_subunit_protein_L10
63	EVVEQEYR*	4/21	16.0%	170083482	glucosephosphate_isomerase
64	VNYGVTVLPTFK*	2/5	28.6%	170083491	maltose_ABC_transporter_periplasmic_substrate- binding_protein
65	YGYQKDQAEK*	9/75	10.7%	170083502	stress_response_protein
66	QQIEEATSDYDREK	17/251	6.3%	170083592	Cpn60_chaperonin_GroEL,_large_subunit_of_GroESL
67	AQIAHFFEHYK	1/30	3.2%	170083670	inorganic_pyrophosphatase

^aHydroxylation sites are denoted in bold, and underline.

^bPTM stoichiometry (%) is the ratio of the number of MS/MS spectra for the peptide bearing the PB-DOPA residue divided by the number of MS/MS spectra for the peptides bearing the corresponding Tyr site, namely the unmodified peptides with or without missing cleavages (cutoff PTMap score > 0.5, FDR < 1%) and modified peptides (cutoff PTMap score > 1.0, FDR < 5%).

^*denotes PB-DOPA peptides with high stoichiometry (≥10%).

Table 2.

List of proteins and peptides bearing PB-DOPA identified in HeLa cell mitochondrial fraction

No	Proteins	IPI#	Sequences a	Stochiometry b
				Spectra # (Modified/Unmodified	%
1	Annexin A2 isoform 1	IPI00418169	TPAQYDASELK	1/42	2.4
2	Electron transfer flavoprotein subunit alpha	IPI00010810	VLVAQHDVYK*	1/9	10.0
3	Inosine-5′-monophosphate dehydrogenase 2	IPI00291510	VSEYAR*	1/7	12.5
4	Glyceraldehyde-3-phosphate dehydrogenase	IPI00219018	YDDIKK	5/71	6.6
			YDNSLK*	2/12	14.3
5	Protein disulfide-isomerase A3	IPI00025252	YKELGEK	3/44	6.4
			LAPEYEAAATR	1/55	1.8
6	Catalase	IPI00465436	LFAYPDTHR	5/62	7.5
7	Superoxide dismutase 2, mitochondrial	IPI00022314	HHAAYVNNLNVTEEK*	2/19	9.5

^aHydroxylation sites are denoted in bold and underline.

^bPTM stoichiometry (%) is the ratio of the number of MS/MS spectra for the peptide bearing the PB-DOPA residue divided by the number of MS/MS spectra for the both peptides bearing the corresponding Tyr site, namely the unmodified peptides with or without missing cleavages (cutoff PTMap score > 0.5, FDR < 1%) and modified peptides (cutoff PTMap score > 1.0, FDR < 5%).

^*denote PB-DOPA peptides with high stoichiometry (≥10%).

Verification of the PB-DOPA sites by HPLC/MS/MS analysis of the recombinant proteins and synthetic peptides

To confirm the in vivo PB-DOPA sites, we expressed and purified two His₆-tagged proteins bearing the PB-DOPA residues in E. coli (Figure 2 and Table 3), superoxide dismutase Fe (GI 170081319) and pyruvate dehydrogenase, decarboxylase component E1 (GI 170079751). The isolated proteins were resolved in SDS-PAGE, followed by in-gel digestion and nano-HPLC/MS/MS analysis. All four peptides bearing PB-DOPA that had been identified from the two proteins in global screening, were confirmed in the corresponding recombinant proteins (Table 3 and Supplementary_figure 3).

Figure 2.

The SDS-PAGE (10-12%) profiles of His₆-tagged protein purification in E. coli. Lanes 1 and 3: molecular weight marker (15-100 kDa), respectively; lane 2, sodB; lane 4, aceE.

Table 3.

Peptides bearing PB-DOPA in the His₆-tagged purified proteins

Gene symbol	Protein name	GI number	Coveragea	Verified peptides bearing PB-DOPAb	Unmodified peptide
sodB	Superoxide dismutase Fe	170081319	52.6%	HHQTYVTNLNNLIK	+
				DALAPHISAETIEYHYGK	+
aceE	Pyruvate dehydrogenase,	170079751	52.6%	EQVAYYKEDEK	+
	Decarboxylase component E1			YLEHR	+

^aThe coverage was calculated by PTMap. PTMap cutoff score is 0.5 for unmodified peptides.

^bHydroxylation sites are denoted in bold and underline.

To further confirm the tyrosine hydroxylation sites in E. coli, we also performed synthetic peptide verification (Figures 3 and 4, and Supplementary_figures 4-6). Two PB-DOPA peptides identified in E. coli lysate were synthesized, namely IAGDY^OHIAK in sugar ABC transporter periplasmic-binding protein, and DALAPHISAETIEY^OHHYGK in superoxide dismutase Fe. The result showed that the MS/MS spectra of the PB-DOPA peptides from E. coli lysate were exactly matched to the MS/MS spectra of their synthetic counterparts (Figure 3A-C and Supplementary_figure 5A-C), and the in vivo PB-DOPA peptides co-eluted with the synthetic counterparts in HPLC (Figure 3D-F and Supplementary_figure 5D-F). We further verified 8 PB-DOPA peptides identified in HeLa mitochondria by MS/MS spectra matching using synthetic peptides (Supplementary_figure 2). Taken together, our results conclusively confirmed the identification of peptides bearing the PB-DOPA residue.

Figure 3.

Identification and verification of the peptide bearing PB-DOPA (IAGDY^OHIAK). Tandem mass spectra (A-C). Tryptic peptide ion from fractionated E. coli lysate (A), synthetic peptide corresponding to the sequences identified in A (B) and mixture of A and B (C). Extracted ion chromatograms of focus ion scans (D-F). Tryptic peptide ion from fractionated E. coli lysate (D), synthetic peptide corresponding to the sequences identified in D (E) and mixture of D and E (F).

Figure 4.

Identification and verification of the peptide bearing PB-DOPA (HHQTY^OHVTNLNNLIK). Tandem mass spectra (A-C). Tryptic peptide ion from His₆-tagged purified protein (A), synthetic peptide corresponding to the sequences identified in A (B) and mixture of A and B (C). Extracted ion chromatograms of focus ion scans (D-F). Tryptic peptide ion from His₆-tagged purified protein (D), synthetic peptide corresponding to the sequences identified in D (E) and mixture of D and E (F).

Evolutionarily conserved PB-DOPA sites in E. coli and human mitochondria

Superoxide dismutase (SOD) is a metal-binding enzyme that catalyzes critical reactions that destroy radicals in the cell. The enzyme is highly redox-sensitive, and involved in catalyzing the dismutation of the superoxide radical anion to prevent the formation of highly aggressive ROS ^31-34. In the present study, we identified Y34 (HHQTY^OHVTNLNNLIK) as one of the two PB-DOPA sites on the E. coli SOD. The modification site was confirmed by MS/MS analysis of the His₆-tagged recombinant protein, and by MS/MS and co-elution experiments of its corresponding synthetic peptide (Figure 4 and Supplementary_figures 7B-C). Previous studies have shown that the Y34 in E. coli SOD is an active site of the enzyme (Supplementary_figure 7A) acting as a proton donor for catalysis ³⁵, promoting heat stability and facilitating substrate binding in both Fe-SODs and Mn-SODs ^{36, 37}. Consequently, it is highly likely that the hydroxylation at Y34 plays a role in regulating its enzymatic activity.

Interestingly, we also identified the PB-DOPA modification at the same residue in human SOD2 from HeLa mitochondria, which is the human ortholog of bacterial SOD. (Supplementary_figure 7A). To verify the identification, we confirmed the MS/MS fragmentation pattern of the PB-DOPA peptide in SOD2 from mitochondria with the corresponding synthetic peptide (Supplementary_figures 7D-E). Previous studies have shown that mutagenesis on Y34, the conserved site in human SOD2, has resulted in decreased enzyme activity ^{32, 33}. Our data therefore revealed an important PB-DOPA site that is evolutionarily conserved, and critical for cellular stress response.

In addition to Y34 in SOD, we also identified an evolutionarily conserved PB-DOPA site on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) – Y256 of GAPDH in E. coli (AATY^OHEQIK) (GI 170081435) (Supplementary_figure 8A) and Y253 of GAPDH in HeLa mitochondrial (Y^OHDDIK) (IPI00219018). GAPDH is an essential enzyme in glycolysis that can catalyze the reaction to convert glyceraldehyde 3-phosphate to D-1,3-bisphospho-glycerate. Accordingly, our data revealed an evolutionarily conserved PB-DOPA site in a protein that is associated with carbohydrate metabolism processes. Identification of the evolutionarily conserved PB-DOPA sites suggests the possibility of a common mechanism that regulates the PTM pathway among a variety of biological species.

We further performed evolution-conservation analysis for all of PB-DOPA sites identified in E. coli lysate among six model species – S. crevisiae, C. elegans, Drosophila, zebra fish, mouse and human. The analysis revealed that among 67 PB-DOPA sites in E. coli, 6 sites were conserved throughout all six species, and 6 sites were conserved in five species (Supplementary_table 2). Among these 12 sites, two sites are from the SOD family and two sites are from GAPDH, including the two PB-DOPA sites that were also identified in HeLa mitochondria analysis.

Structural analysis of PB-DOPA sites in E. coli

Protein phosphorylation has been shown to preferably occur in the unstructured regions, while lysine acetylation is found in structured motifs ^{38, 39}. We studied the secondary structure characteristics of PB-DOPA sites identified in the E. coli lysate, and compared them to the secondary structure characteristics of all Tyr sites in E. coli proteins. Our data showed that PB-DOPA is enriched on the helix structure (p=0.0012) and significantly less represented on the turn structure (p=0.012) comparing to all the tyrosine residues (Figure 5A). Hence, PB-DOPA modification, similar to lysine acetylation, is likely to be enriched in the structured sequences and less abundant in the unstructured regions.

Figure 5.

Bioinformatic analysis. Secondary structure analysis (A) sequence logo analysis of PB-DOPA sites (B) and enrichment analysis (C) in the biological process in proteins bearing PB-DOPA in E. coli.

We further performed sequence logo analysis for the PB-DOPA sites identified in the E. coli lysate (Figure 5B) ⁴⁰, and compared the abundance of different amino acids on each flanking position of the PB-DOPA site with that of all Tyr residues in E. coli proteins from the database. We found that the positively charged amino acid Lys was enriched in several positions including +9, −5, −1 and +10, while negatively charged amino acid Glu was enriched in +2 and +3 positions.

Biological process enrichment analysis

To elucidate cellular pathways that may be associated with PB-DOPA, we performed enrichment analysis of the biological processes on the identified PB-DOPA proteins by comparing the Gene Ontology (GO) annotations of the PB-DOPA proteins and E. coli proteome (Table 1 and Supplementary_table 1). Our data revealed that proteins bearing PB-DOPA were dominantly enriched in carbohydrate metabolism such as glycolysis and glucogenesis (Figure 5C). Among the 27 biological processes with enrichment p value less than 0.01, 14 processes were involved with carbohydrate metabolism, and all the enriched processes were involved with energy metabolism in the cell.

DISCUSSION

Identification of the PTM substrate proteins and their PTM sites are typically the first steps toward dissection of the PTM pathways, and understanding their biological functions. The protein substrates of tyrosine hydroxylation cannot easily be identified by conventional isotopic labeling. Additionally, there are still no modification-specific pan antibodies, such as those for phosphotyrosine and lysine acetylation, to detect and confirm its PTM peptides. Accordingly, biology studies of tyrosine hydroxylation have previously proceeded very slowly.

To address this problem, we carried out the first proteomics screening to identify PB-DOPA proteins. The study described here identified 67 novel sites of PB-DOPA in 43 proteins in E. coli, and 9 novel PB-DOPA sites in 7 proteins from HeLa mitochondria. Our approach involves unrestrictive sequence alignment using PTMap software developed in-house. Unlike conventional database searching software, PTMap requires exclusive modification-site localization for all unrestrictive PTM identifications. Given that hydroxylation or oxidation can occur on various amino acids (http://www.unimod.org), this feature of PTMap offers unique advantage by removing ambiguous PTM identifications with oxidation on amino acids other than Tyr.

A total of 4,715 and 3,012 Tyr residues were identified from 12,184 non-redundant E. coli peptides and 8,151 HeLa mitochondrial peptides, respectively. These results allow us to estimate the relative abundance of PB-DOPA to be ~ 1.4% and ~ 0.3% in E. coli and HeLa mitochondria, respectively. The result was a surprise to us, as mitochondria is the major cellular organelle that generates radicals and other reactive oxygen species (ROS). The much lower abundance of PB-DOPA in mitochondria than in E. coli could suggest that either ROS may not be the major cause of PB-DOPA in E. coli, or the mechanisms to prevent oxidative damage in mammalian cells are much better developed than E. coli.

Our results suggest that PB-DOPA is likely involved in the regulation of SOD2 activity. The same PB-DOPA site was found in SOD2 protein from both E. coli and HeLa mitochondria. Structure analysis showed that PB-DOPA preferred structured protein segments to unstructured regions. More importantly, proteins bearing PB-DOPA were found to be highly enriched in the pathways involved in cellular energy metabolism and especially carbohydrate metabolism. Our data therefore indicate the possibility of intrinsic cellular mechanism that regulates the PB-DOPA pathway. Finally, to our knowledge, this is the first study demonstrating that the PB-DOPA is present in prokaryotic cells and eukaryotic mitochondria.

Hydroxylation of proline and lysine and their regulatory enzymes such as prolyl hydroxylases are known to contribute to a variety of physiolgical and pathophysiological processes, such as tumorigenesis, erythropoiesis, angiogenesis ⁴¹. Hydroxylation at the tyrosine residue changes its structure. It is anticipated that such change is likely to induce alteration of structure and functions for a portion of its substrates, similar to proline hydroxylation (e.g., HIFα). Furthermore the alteration of protein structure can induce the preference of degradation in biological pathway ^{11, 42}.

Many questions remain in the field of tyrosine hydroxylation. Based on the information obtained in this study, PB-DOPA is an abundant protein post-translational modification. It is highly likely that multiple enzymes remain to be identified in the cells. Is the PB-DOPA status dynamically changed between cells in different organs, cell lines, developmental stages, and environmental conditions? What are the main factors that determine the levels of PB-DOPA? What are the major functions of PB-DOPA in cellular functions and pathological conditions? The PB-DOPA substrates identified in this study could drive experimental efforts toward functional characterization of the PB-DOPA proteins and the anatomy of the PB-DOPA pathways.