. 2019 Sep 10;8:e46490. doi: 10.7554/eLife.46490

Table 2. Summary of oxidations observed on protein substrates produced by IVTT and reacted with the indicated PHD enzyme(s).

Mass spectrometry was performed on IVTT-derived substrates (column 1) reacted in the presence or absence of the indicated PHD isoform (column 2). Substrates were immunopurified by FLAG affinity and digested with the specified protease(s) (column 3) to yield peptides encompassing the putative target prolyl site, which are indicated in bold (column 4); note, during sample processing peptides containing cysteine residues were derivatized with iodoacetamide to give carbamidomethylated cysteine (+57.02). Reference to primary data for non-HIF substrates (i.e., MSMS assignment and quantitation) is provided in column five and summarised in columns 6–10. Assigned oxidations are listed in column 6; no oxidation detected (ND); oxidation detected but not localised to a specific residue (NL). Note, prolyl hydroxylation was not detected. Quantitative data for control and PHD-reacted IVTTs is given for these oxidations (columns 7–8, respectively). Low abundance peptide ions of compatible mass for oxidation, which were below the threshold for MSMS determination but present in LC-MS data (within −5 min retention time window), were also quantified (columns 9–10). The abundance of each assigned or putative oxidation is expressed as a percentage of the non-hydroxylated peptide. Summary results for the products of control reactions that were conducted in parallel on HIF-1α to verify PHD enzyme activity are shown in columns 11–14; values were obtained by similar methods (see Supplementary file 2 for figures depicting these primary data). Each row relates a specific ‘test’ reaction to its batch control HIF-1α reaction; note that high levels of activity on HIF-1α substrates were observed upon addition of exogenous PHD enzyme in all controls.

Table 2—source data 1. Peptide standards employed in IVTT hydroxylation assays.

The table lists synthetic peptide sequences corresponding to unoxidised and hydroxylated variants (column 3) of protease-digested peptides assigned and quantified in IVTT hydroxylation assays. Equimolar injections of the indicated peptide variants were used for comparison of detection efficiency (column 4) and chromatographic elution time (column 5) by LC-MSMS analysis. References to the primary XIC data are indicated in column 6.

elife-46490-table2-data1.xlsx^{(32.9KB, xlsx)}

DOI: 10.7554/eLife.46490.008

			Non-HIF substrate							HIF-1α control
			Quantified peptide	Figure	Oxidation	Assigned ox (%)		Unassigned ions (%)		HIF-1α P402ox (%)		HIF-1α P564ox (%)
Substrate	PHD Isoform	Protease	Quantified peptide	Figure	Oxidation	Control	Enzyme	Control	Enzyme	Control	Enzyme	Control	Enzyme
ACACB	PHD3	LysC	RIPVQAVWAGWGHASENPKLPELLC(+57.02)K	3, s1	ND	-	-	0.4%	1.7%	58%	52%	53%	72%
ACACB	PHD3	LysC	DVDEGLEAAERIGFPLMIK	3, s2	M452	76%	77%	2.3%	0.9%	58%	52%	53%	72%
ACTB	PHD3	Trypsin	DLYANTVLSGGTTMYPGIADR	3, s3	M305	71%	48%	ND	ND	6.3%	9.9%	29%	91%
ACTB	PHD3	Trypsin	EITALAPSTMK	3, s4	M325	54%	30%	0.3%	0.1%	6.3%	9.9%	29%	91%
ADRB2	PHD3	Trypsin	LLC(+57.02)EDLPGTEDFVGHQGTVPSDNIDSQGR	3, s5	D380	2.8%	1.5%	ND	<0.1%	6.3%	9.9%	29%	91%
AKT1	PHD2	Trypsin	SGSPSDNSGAEEMEVSLAK	3, s6	M134	35%	39%	ND	ND	18%	94%	30%	96%
			TFC(+57.02)GTPEYLAPEVLEDNDYGR	3, s7	ox: NL	0.6%	0.8%	ND	ND
			LSPPFKPQVTSETDTR	3, s8	ND	-	-	ND	ND
ATF4	PHD3	Elastase	GHLPESLTKPDQVAPFTFLQPLPLSPG	3, s9	ND	-	-	ND	ND	6.3%	9.9%	29%	91%
ATF4	PHD3	Elastase	STPDHSFSLELGSEVDITEGDRKPDYT	3, s10	ND	-	-	ND	ND	6.3%	9.9%	29%	91%
CENPN	PHD2	Trypsin	SLAPAGIADAPLSPLLTC(+57.02)IPNKR	3, s11	ND	-	-	ND	ND	ND	ND	22%	94%
CEP192	PHD1	Trypsin	WHLSSLAPPYVK	3, s12	ND	-	-	<0.1%	ND	51%	95%	60%	82%
EEF2K	PHD2	Trypsin	HMPDPWAEFHLEDIATER	3, s13	M95	48%	38%	ND	0.2%	0.5%	88%	36%	93%
EPOR	PHD3	LysC + GluC	YTILDPSSQLLRPWTLC(+57.02)PELPPTPPHLK	3, s14	ox: NL	1.4%	1.4%	ND	ND	9.5%	15%	23%	94%
EPOR	PHD3	LysC + GluC	YTILDPSSQLLRPWTLC(+57.02)PELPPTPPHLK	3, s14	diox: W439	2.0%	1.8%	ND	ND	9.5%	15%	23%	94%
FLNA	PHD2	Trypsin	FNEEHIPDSPFVVPVASPSGDAR	3, s15	ND	-	-	0.1%	0.3%	ND	ND	22%	94%
FOXO3	PHD1	Trypsin	GSGLGSPTSSFNSTVFGPSSLNSLR	3, s16	ND	-	-	2.2%	0.9%	14%	97%	25%	86%
IKBKB	PHD1	Trypsin	ELDQGSLC(+57.02)TSFVGTLQYLAPELLEQQK	3, s17	ND	-	-	14%	14%	14%	97%	25%	86%
MAPK6	PHD3	Trypsin	YMDLKPLGC(+57.02)GGNGLVFSAVDNDC(+57.02)DKR	3	M21	19%	12%	0.3%	0.3%	9.5%	15%	23%	94%
NDRG3	PHD2	Trypsin	MADC(+57.02)GGLPQVVQPGK	3, s18	M287	48%	21%	1.8%	0.9%	17%	92%	24%	93%
PDE4D	PHD2	Trypsin	LMHSSSLTNSSIPR	3, s19	M371	61%	36%	ND	ND	0.5%	88%	36%	93%
PDE4D	PHD2	Trypsin	IAELSGNRPLTVIMHTIFQER	3, s20	M424	51%	45%	ND	ND	0.5%	88%	36%	93%
PKM	PHD3	Trypsin	LAPITSDPTEATAVGAVEASFK	3, s21	ND	-	-	<0.1%	0.1%	6.3%	9.9%	29%	91%
PPP2R2A	PHD2	Trypsin	IWDLNMENRPVETYQVHEYLR	3, s22	M315	14%	20%	ND	ND	27%	86%	36%	91%
SPRY2	PHD1	Trypsin	AQSGNGSQPLLQTPR	-	-	-	-	ND	ND	-	-	-	-
	PHD3		AQSGNGSQPLLQTPR	3, s23	ND	-	-	ND	ND	ND	ND	52%	92%
	PHD1		LLGSSFSSGPVADGIIR	3, s24	ND	-	-	0.9%	ND	9.5%	92%	23%	83%
	PHD3		LLGSSFSSGPVADGIIR	3, s25	ND	-	-	ND	ND	ND	ND	52%	92%
	PHD1		SELKPGELKPLSK	3, s26	ND	-	-	ND	ND	9.5%	92%	23%	83%
	PHD3		SELKPGELKPLSK	3, s27	ND	-	-	ND	ND	ND	ND	52%	92%
TELO2	PHD3	Trypsin	AVLIC(+57.02)LAQLGEPELR	3, s28	ND	-	-	8.9%	6.5%	ND	ND	52%	92%
THRA	PHD2	Trypsin	SLQQRPEPTPEEWDLIHIATEAHR	3, s29	ox: NL	0.9%	0.7%	1.8%	1.3%	15%	94%	26%	96%
	PHD2			3, s29	diox: W165	5.4%	5.1%	1.8%	1.3%	15%	94%	26%	96%
	PHD3			3, s30	ox: NL	0.9%	0.8%	1.8%	0.8%	15%	16%	26%	86%
	PHD3			3, s30	diox: W165	5.4%	4.8%	1.8%	0.8%	15%	16%	26%	86%
TP53	PHD1	Trypsin	TC(+57.02)PVQLWVDSTPPPGTR	3, s31	ND	-	-	ND	ND	9.5%	92%	23%	83%
TP53	PHD3	LysC	EPGGSRAHSSHLK	3, s32	ND	-	-	ND	0.4%	21%	24%	30%	78%
TRPA1	PHD2	Trypsin	NLRPEFMQMQQIK	3, s33	M397	31%	30%	ND	ND	0.5%	88%	36%	93%
TRPA1	PHD2	Trypsin	NLRPEFMQMQQIK	3, s33	M399	33%	34%	ND	ND	0.5%	88%	36%	93%