J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 20, December 2022, pages 40-55

Metabolic fate of peptides in a rice protein hydrolysate in rat intestine and blood after oral administration

Figure 1. Mass chromatogram of AccQ-derivatives of SEC Fr. 37 of RPH in precursor ion scan mode (upper panel) and spectra of the product ions from precursor ions in peaks 18, 15, and 19, respectively (lower panels). The product ion scan was carried out using collision energies (CEs) at −35 and −15 eV. The immonium ions of amino acids are indicated as one-letter abbreviations with asterisk.

Figure 2. Mass chromatogram of a pyroglutamyl peptide of SEC Fr. 37 of RPH in precursor ion scan mode (upper panel) and spectra of product ions from the precursor ion in peak 4 (lower panels). The product ion scan was carried out using collision energies (CEs) at −35 and −15 eV. The immonium ions of amino acids are indicated as one letter abbreviation with asterisk. Pyroglutamyl residue is represented as pE. Immonium ions and related ions derived from pyroglutamic acid are indicated as pE with one and two asterisks, respectively.

Figure 3. Concentrations of peptides in RPH and its exopeptidase digest. The amino acid residues are indicated as one letter abbreviations, and pE represents pyroglutamyl residue.

Figure 4. Isomers of di-peptides with aspartyl residue at the amino terminus in RPH and its exopeptidase digest. Four isomers of the Asp-X-type peptides (L-α-Asp-X, D-α-Asp-X, L-β-Asp-X, and D-β-Asp-X) were synthesized. The AccQ derivatives of synthetic peptides (std.) and peptides in RPH and its exopeptidase digest were analyzed using LC-MS/MS. Resolution of isomers of Asp-Phe, Asp-Ile/Leu, Asp-Glu, and Asp-Val by LC-MS/MS are shown in Figure 4a–d, respectively. Lα, Lβ, Dα, and Dβ represent peptides containing L-α-aspartyl residue, L-β-aspartyl residue, D-α-aspartyl residue, and D-β-aspartyl residue, respectively. Resolution between L-α-Asp-Ile and L-α-Asp-Leu, and between D-α-Asp-Ile and D-α-Asp-Leu, could not be performed under present condition.

Figure 5. Amounts and concentrations of RPH-derived peptides containing amino group in rat (a) lumen, (b) ileal tissue, and (c) portal blood plasma before and after administration of RPH. These data are presented with peptide concentrations in RPH and its exopeptidase digest (d). Data are shown as mean ± standard deviation. (a–c, n = 3). Asterisks indicate that significant differences between values before and after administration of RPH as determined by t-test (*p < 0.05, **p < 0.01).

Figure 6. Amounts and concentrations of RPH-derived pyroglutamyl peptides in rat (a) lumen, (b) ileal tissue, and (c) portal blood plasma before and after administration of RPH. These data are presented with pyroglutamyl peptide concentrations in RPH and its exopeptidase digest (d). Data are shown as mean ± standard deviation. (a–c, n = 3). Asterisks indicate that significant differences between values before and after administration of RPH as determined by t-test (*p < 0.05, **p < 0.01).

**Table 1.** Summary of estimated peptide sequences of AccQ-derivatives in RPH
Peak No.	SEC Fr.	Estimated peptide sequence	Precursor ions (m/z)	Product ions (m/z)
¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides. ²The identities of the tentatively assigned peptides were not confirmed, as their ion intensity was low in single ion monitoring mode. Supplemental Figure 1 shows the mass chromatograms with peak numbers.
1	36	AS	347	170.9 (AccQ, b1), 176.9 (y1), 213.9 (a2), 242.0 (b2)
2	36	SA	347	60.5 (Ser*), 171.0 (AccQ, b1), 177.2 (y2), 257.8 (b2)
3	36, 37	AA	331	161.0 (y2), 171.0 (AccQ, b1), 241.8 (b2)
4	36	GQ	374	84.1 (Gln**), 146.7 (y1), 170.9 (AccQ, b1), 203.7 (y2), 227.8 (b2)
5	36	AN	374	44.4 (Ala*), 132.9 (y1), 170.9 (AccQ, b1), 203.9 (y2), 214.1 (a2), 241.7 (b2)
6	36	AQ	388	44.5 (Ala*), 147.0 (y1), 170.9 (AccQ, b1), 213.9 (y2), 217.9 (y2), 241.9 (b2)
7	36	AT	361	44.0 (Ala*), 119.7 (y1), 171.0 (AccQ, b1), 190.8 (y2), 213.7 (a2), 242.1 (b2)
8	36	AE	389	148.1 (y1), 170.9 (AccQ, b1), 213.9 (a2), 219.0 (y2), 242.1 (b2)
9	36	TA	361	170.9 (AccQ, b1), 190.7 (y2), 272.1 (b2)
10	36	PA	357	70.1 (Pro*), 171.0 (AccQ, b1), 186.9 (y2), 240.0 (a2)
11	36	VS	375	72.3 (Val*), 171.0 (AccQ, b1), 205.0 (y2), 242.0 (a2), 270.2 (b2)
12	36	VT	389	72.2 (Val*), 170.9 (AccQ, b1), 218.8 (y2), 241.9 (a2), 269.8 (b2)
13	36	VA	359	72.2 (Val*), 170.9 (AccQ, b1), 189.0 (y2), 242.0 (a2), 269.9 (b2)
14	36	IS, LS¹	389	86.1 (Ile/Leu*), 171.0 (AccQ, b1), 219.0 (y2), 256.0 (a2), 283.6 (b2)
15	36, 37	SI, SL¹	389	171.0 (AccQ, b1), 219.0 (y2), 258.0 (b2)
16	36	IA, LA¹	373	86.1 (Ile/Leu*), 170.9 (AccQ, b1), 203.0 (y2), 255.9 (a2), 283.9 (b2)
17	36	VV	387	72.4 (Val*), 170.8 (AccQ, b1), 217.0 (y2), 242.1 (a2), 270.3 (b2)
18	37	GI, GL¹	359	170.9 (AccQ, b1), 188.9 (y2), 227.9 (b2)
19	37	AI, AL¹	373	132.1 (y1), 170.9 (AccQ, b1), 203.1 (y2), 213.9 (a2), 241.9 (b2)
20	42–45	GF	393	171.0 (AccQ, b1), 227.9 (b2)
21	36, 37	SQ	404	60.2 (Ser), 83.6 (Gln*), 147.0 (y1), 170.9 (AccQ, b1), 233.8 (y2), 230.0 (a2), 257.8 (b2)
22	36	DT²	405	74.2 (Thr*), 171.1 (AccQ, b1), 234.7 (y2), 258.2 (a2)
23	36	EQ²	446	83.8 (Glu, Gln), 147.2 (y1), 171.1 (AccQ, b1), 272.3 (a2), 275.5 (y2), 299.6 (b2)
24	36, 37	TE	419	74.4 (Thr), 84.4 (Glu*), 147.7 (y1), 170.8 (AccQ, b1), 243.7 (a2), 248.7 (y2)
25	36	GVS	432	72.1 (Val*), 106.1 (y1), 170.9 (AccQ, b1), 199.9 (a2), 228.1 (b2), 327.1 (b3), 385.6 (a4)
26	36	GVA	416	72.4 (Val*), 90.1 (y1), 170.9 (AccQ, b1), 188.8 (y2), 200.0 (a2), 228.1 (b2), 327.0 (b3)
27	36	VQ	416	71.9 (Val), 101.1 (Gln), 147.1 (y1), 171.0 (AccQ, b1), 242.1 (a2), 245.8 (y2), 269.8 (b2)
28	36	GIS, GLS²	446	86.3 (Ile/Leu*), 106.1 (y1), 171.0 (AccQ, b1), 199.7 (a2), 228.0 (b2), 276.0 (y3), 340.6 (b3)
29	36, 37	VE	417	72.0 (Val*), 148.2 (y1), 170.9 (AccQ, b1), 242.1 (a1), 246.9 (y2), 270.1 (b2)
30	36	GAI, GAL²	430	44.3 (Ala), 86.3 (Ile/Leu), 132.1 (y1), 171.2 (AccQ, b1), 228.1 (b2), 259.8 (y2)
31	36	IQ, LQ¹	430	84.1 (Gln*), 86.2 (Ile/Leu), 146.9 (y1), 170.8 (AccQ, b1), 256.1 (a2), 259.8 (y2), 283.9 (b2)
32	36	IT, LT²	403	86.2 (Ile/Leu*), 119.7 (y1), 171.0 (AccQ, b1), 232.5 (y2), 255.9 (a2)
33	36, 37	TI, TL¹	403	86.1 (Ile/Leu*), 170.9 (AccQ, b1), 233.0 (y2), 272.0 (b2)
34	36, 37	IV, LV¹	401	86.0 (Ile/Leu*), 171.1 (AccQ, b1), 230.9 (y2), 255.8 (a2), 284.0 (b2)
35	36, 37	VI, VL¹	401	72.1 (Val*), 170.8 (AccQ, b1), 231.1 (y2), 242.3 (a2), 269.8 (b2)
36	36, 37	MI, ML¹	433	86.2 (ile/Leu), 104.2 (Met), 171.0 (AccQ, b1), 274.0 (a2)
37	36, 37	II, IL, LI, LL¹	415	86.2 (Ile/Leu*), 171.0 (AccQ, b1), 244.9 (y2), 284.1 (b2)
38	37	NE²	432	87.7 (Asn), 101.7 (Glu), 171.0 (AccQ, b1), 256.9 (a2), 261.9 (y2), 285.0 (b2)
39	37	SE	405	60.4 (Ser*), 147.8 (y1), 171.0 (AccQ, b1), 234.9 (y2)
40	37	SM²	407	150.0 (y1), 170.9 (AccQ, b1), 236.9 (y2), 257.7 (b2)
41	37	NI, NL¹	416	87.0 (Asn*), 132.0 (y1), 171.0 (AccQ, b1), 245.9 (y2), 256.8 (a2), 284.9 (b2)
42	37	DI, DL¹	417	86.3 (Ile/Leu*), 131.7 (y1), 170.9 (AccQ, b1), 247.1 (y2), 286.0 (b2)
43	37	EI, EL¹	431	84.1 (Glu*), 86.2 (Ile/Leu), 132.4 (y1), 170.8 (AccQ, b1), 261.0 (y2), 271.9 (a2), 299.8 (b2)
44	37	IM, LM²	433	86.3 (Ile/Leu*), 170.8 (AccQ, b1), 256.1 (a2), 263.1 (y2), 284.3 (b2)
45	41–43	SF	423	119.9 (Phe*), 166.0 (y1), 171.0 (AccQ, b1), 230.0 (a2), 253.0 (y2), 258.0 (b2)
46	41, 42	TF	437	74.2 (Thr*), 166.0 (y1), 170.9 (AccQ, b1), 243.7 (a2), 267.0 (y2), 271.9 (b2)
47	41–43	AF	407	120.1 (Phe*), 165.9 (y1), 171.0 (AccQ, b1), 213.8 (a2), 236.9 (y2), 241.9 (b2)
48	41–43	VF	435	72.1(Val*), 170.9 (AccQ, b1), 242.0 (a2), 264.9 (y2), 270.0 (b2)
49	42–45	IF, LF¹	449	86.2 (Ile/Leu), 119.5 (Phe), 171.0 (AccQ, b1), 256.1 (a2), 279.1 (y2), 283.8 (b2)
50	43	SY	439	60.4 (Ser), 136.1 (Tyr), 171.0 (AccQ, b1), 182.1 (y1), 229.7 (a2), 257.9 (b2), 269.1 (y2)
51	43	AY	423	171.0 (AccQ, b1), 182.0 (y1), 213.8 (a2), 241.9 (b2)
52	36	STS	464	59.7 (Ser*), 106.1 (y1), 171.2 (AccQ, b1), 219.9 (a2), 257.6 (b2)
53	36. 37	GVE²	474	148.0 (y1), 170.8 (AccQ, b1), 227.9 (b2), 303.6 (y3)
54	36, 37	GIN, GLN²	473	85.8 (Ile/Leu), 87.3 (Asn), 133.1 (y1), 171.1 (AccQ, b1), 199.5 (a2), 227.8 (b2), 303.1 (y3)
55	36	GIT, GLT²	460	85.9 (Ile/Leu*), 119.8 (y1), 170.9 (AccQ, b1), 199.6 (a2), 228.1 (b2), 290.1 (y3), 340.6 (b3)
56	36	ASI, ASL¹	460	44.1 (Ala), 59.6 (Ser), 85.7 (Ile/Leu*), 171.0 (AccQ, b1), 241.4 (b2), 289.5 (y3), 329.2 (b3)
57	36	AVI, AVL¹	472	44.4 (Ala), 71.9 (Val), 170.4 (AccQ, b1), 214.1 (a2), 241.7 (b2), 301.7 (y3), 340.9 (b3)
58	36	IAI, IAL, LAI, LAL²	486	86.2 (Ile/Leu*), 170.7 (AccQ, b1), 283.7 (b2), 315.9 (y3), 355.3 (b3)
59	37–41	SGF	480	60.6 (Ser), 120.5 (Phe), 165.9 (y1), 170.4 (AccQ, b1), 222.8 (y2), 258.2 (b2)
60	37, 38	GTF²	494	73.9 (Thr), 119.7 (Phe), 165.8 (y1), 227.7 (b2), 267.1 (y2), 300.7 (a3), 323.7 (y3)
61	37–40	GAF²	464	120.0 (Phe*), 166.0 (y1), 170.8 (AccQ, b1), 227.9 (b2)
62	37–42	FQ²	464	84.5 (Gln*), 119.9 (Phe), 147.4 (y1), 170.7 (AccQ, b1), 289.6 (a2), 318.2 (b2)
63	37	AAF²	478	166.0 (y1), 170.8 (AccQ, b1), 241.8 (b2), 307.7 (y3)
64	37	GII, GIL, GLI, GLL²	472	86.0 (Ile/Leu*), 131.9 (y1), 170.7 (AccQ, b1), 200.2 (a2), 227.9 (b2), 340.8 (b3)
65	41	GGF²	450	120.1 (Phe*), 165.9 (y1), 171.1 (AccQ, b1), 199.7 (a2), 227.8 (b2), 279.8 (y3), 285.0 (b3)
66	41	DF	451	88.2 (Asp*), 171.0 (AccQ, b1), 257.7 (a2), 280.7 (y2), 285.9 (b2)
67	41	FE²	465	119.9 (Phe*), 147.9 (y1), 171.0 (AccQ, b1), 294.6 (y2), 317.6 (b2)
68	41, 42	GVF²	492	72.0 (Val), 119.4 (Phe), 166.0 (y1), 171.0 (AccQ, b1), 199.8 (a2), 227.9 (b2), 326.5 (b3)
69	42	YQ²	480	83.8 (Gln*), 100.7 (Gln), 136.0 (Tyr*), 147.0 (y1), 170.8 (AccQ, b1), 305.9 (a2), 333.6 (b2)
70	42–44	NF²	450	86.6 (Asn*), 165.8 (y1), 170.8 (AccQ, b1), 284.9 (b2)
71	42–44	EF²	465	84.0 (Glu*), 102.1 (Glu), 120.2 (Phe*), 165.9 (y1), 171.0 (AccQ, b1), 299.8 (b2)
72	43–45	TY	453	74.0 (Thr), 136.0 (Tyr), 271.8 (b2), 282.7 (y2)
73	44, 45	VY²	451	72.3 (Val*), 170.9 (AccQ, b1), 241.9 (a2), 269.8 (b2), 280.6 (y2)
74	36	STE²	506	59.9 (Ser), 74.1 (Thr), 147.6 (y1), 170.9 (AccQ, b1), 229.5 (a2), 257.7 (b2), 335.6 (y3)
75	36	LTE, ITE²	532	85.8 (Ile/Leu*), 147.7 (y1), 170.9 (AccQ, b1), 255.5 (a2), 361.6 (y3), 385.2 (b3)
76	36	TPI, TPL¹	500	69.7 (Pro), 85.7 (Ile/Leu), 132.2 (y1), 170.5 (AccQ, b1), 228.8 (y2), 271.8 (b2), 330.1 (y3)
77	36	IIQ, ILQ, LIQ, LLQ¹	543	86.1 (Ile/Leu), 100.8 (Gln), 147.1 (y1), 171.0 (AccQ, b1), 256.0 (a2), 284.0 (b2), 372.9 (y3), 396.9 (b3)
78	37	DVI, DVL¹	516	85.9 (Ile/Leu*), 171.0 (AccQ, b1), 285.6 (b2), 384.9 (b3)
79	37, 38	IAF, LAF¹	520	86.0 (Ile/Leu*), 165.6 (y1), 171.0 (AccQ, b1), 236.4 (y2), 256.1 (a2), 283.8 (b2), 354.7 (b3)
80	41–44	NIF, NLF¹	563	86.1 (Ile/Leu), 119.7 (Phe), 170.8 (AccQ, b1), 257.1 (a2), 284.9 (b2), 397.8 (b3)
81	41–44	SIF, SLF¹	536	60.2 (Ser), 86.1 (Ile/Leu), 119.9 (Phe*), 165.6 (y1), 170.9 (AccQ, b1), 229.7 (a2), 371.0 (b3)

Table 1. Summary of estimated peptide sequences of AccQ-derivatives in RPH

Peak No.

SEC Fr.

Estimated peptide sequence

Precursor ions (m/z)

Product ions (m/z)

¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides. ²The identities of the tentatively assigned peptides were not confirmed, as their ion intensity was low in single ion monitoring mode. Supplemental Figure 1 shows the mass chromatograms with peak numbers.

347

170.9 (AccQ, b1), 176.9 (y1), 213.9 (a2), 242.0 (b2)

347

60.5 (Ser*), 171.0 (AccQ, b1), 177.2 (y2), 257.8 (b2)

36, 37

331

161.0 (y2), 171.0 (AccQ, b1), 241.8 (b2)

374

84.1 (Gln**), 146.7 (y1), 170.9 (AccQ, b1), 203.7 (y2), 227.8 (b2)

374

44.4 (Ala*), 132.9 (y1), 170.9 (AccQ, b1), 203.9 (y2), 214.1 (a2), 241.7 (b2)

388

44.5 (Ala*), 147.0 (y1), 170.9 (AccQ, b1), 213.9 (y2), 217.9 (y2), 241.9 (b2)

361

44.0 (Ala*), 119.7 (y1), 171.0 (AccQ, b1), 190.8 (y2), 213.7 (a2), 242.1 (b2)

389

148.1 (y1), 170.9 (AccQ, b1), 213.9 (a2), 219.0 (y2), 242.1 (b2)

361

170.9 (AccQ, b1), 190.7 (y2), 272.1 (b2)

357

70.1 (Pro*), 171.0 (AccQ, b1), 186.9 (y2), 240.0 (a2)

375

72.3 (Val*), 171.0 (AccQ, b1), 205.0 (y2), 242.0 (a2), 270.2 (b2)

389

72.2 (Val*), 170.9 (AccQ, b1), 218.8 (y2), 241.9 (a2), 269.8 (b2)

359

72.2 (Val*), 170.9 (AccQ, b1), 189.0 (y2), 242.0 (a2), 269.9 (b2)

IS, LS¹

389

86.1 (Ile/Leu*), 171.0 (AccQ, b1), 219.0 (y2), 256.0 (a2), 283.6 (b2)

36, 37

SI, SL¹

389

171.0 (AccQ, b1), 219.0 (y2), 258.0 (b2)

IA, LA¹

373

86.1 (Ile/Leu*), 170.9 (AccQ, b1), 203.0 (y2), 255.9 (a2), 283.9 (b2)

387

72.4 (Val*), 170.8 (AccQ, b1), 217.0 (y2), 242.1 (a2), 270.3 (b2)

GI, GL¹

359

170.9 (AccQ, b1), 188.9 (y2), 227.9 (b2)

AI, AL¹

373

132.1 (y1), 170.9 (AccQ, b1), 203.1 (y2), 213.9 (a2), 241.9 (b2)

42–45

393

171.0 (AccQ, b1), 227.9 (b2)

36, 37

404

60.2 (Ser*), 83.6 (Gln**), 147.0 (y1), 170.9 (AccQ, b1), 233.8 (y2), 230.0 (a2), 257.8 (b2)

DT²

405

74.2 (Thr*), 171.1 (AccQ, b1), 234.7 (y2), 258.2 (a2)

EQ²

446

83.8 (Glu**, Gln**), 147.2 (y1), 171.1 (AccQ, b1), 272.3 (a2), 275.5 (y2), 299.6 (b2)

36, 37

419

74.4 (Thr*), 84.4 (Glu**), 147.7 (y1), 170.8 (AccQ, b1), 243.7 (a2), 248.7 (y2)

GVS

432

72.1 (Val*), 106.1 (y1), 170.9 (AccQ, b1), 199.9 (a2), 228.1 (b2), 327.1 (b3), 385.6 (a4)

GVA

416

72.4 (Val*), 90.1 (y1), 170.9 (AccQ, b1), 188.8 (y2), 200.0 (a2), 228.1 (b2), 327.0 (b3)

416

71.9 (Val*), 101.1 (Gln*), 147.1 (y1), 171.0 (AccQ, b1), 242.1 (a2), 245.8 (y2), 269.8 (b2)

GIS, GLS²

446

86.3 (Ile/Leu*), 106.1 (y1), 171.0 (AccQ, b1), 199.7 (a2), 228.0 (b2), 276.0 (y3), 340.6 (b3)

36, 37

417

72.0 (Val*), 148.2 (y1), 170.9 (AccQ, b1), 242.1 (a1), 246.9 (y2), 270.1 (b2)

GAI, GAL²

430

44.3 (Ala*), 86.3 (Ile/Leu*), 132.1 (y1), 171.2 (AccQ, b1), 228.1 (b2), 259.8 (y2)

IQ, LQ¹

430

84.1 (Gln**), 86.2 (Ile/Leu*), 146.9 (y1), 170.8 (AccQ, b1), 256.1 (a2), 259.8 (y2), 283.9 (b2)

IT, LT²

403

86.2 (Ile/Leu*), 119.7 (y1), 171.0 (AccQ, b1), 232.5 (y2), 255.9 (a2)

36, 37

TI, TL¹

403

86.1 (Ile/Leu*), 170.9 (AccQ, b1), 233.0 (y2), 272.0 (b2)

36, 37

IV, LV¹

401

86.0 (Ile/Leu*), 171.1 (AccQ, b1), 230.9 (y2), 255.8 (a2), 284.0 (b2)

36, 37

VI, VL¹

401

72.1 (Val*), 170.8 (AccQ, b1), 231.1 (y2), 242.3 (a2), 269.8 (b2)

36, 37

MI, ML¹

433

86.2 (ile/Leu*), 104.2 (Met*), 171.0 (AccQ, b1), 274.0 (a2)

36, 37

II, IL, LI, LL¹

415

86.2 (Ile/Leu*), 171.0 (AccQ, b1), 244.9 (y2), 284.1 (b2)

NE²

432

87.7 (Asn*), 101.7 (Glu*), 171.0 (AccQ, b1), 256.9 (a2), 261.9 (y2), 285.0 (b2)

405

60.4 (Ser*), 147.8 (y1), 171.0 (AccQ, b1), 234.9 (y2)

SM²

407

150.0 (y1), 170.9 (AccQ, b1), 236.9 (y2), 257.7 (b2)

NI, NL¹

416

87.0 (Asn*), 132.0 (y1), 171.0 (AccQ, b1), 245.9 (y2), 256.8 (a2), 284.9 (b2)

DI, DL¹

417

86.3 (Ile/Leu*), 131.7 (y1), 170.9 (AccQ, b1), 247.1 (y2), 286.0 (b2)

EI, EL¹

431

84.1 (Glu**), 86.2 (Ile/Leu*), 132.4 (y1), 170.8 (AccQ, b1), 261.0 (y2), 271.9 (a2), 299.8 (b2)

IM, LM²

433

86.3 (Ile/Leu*), 170.8 (AccQ, b1), 256.1 (a2), 263.1 (y2), 284.3 (b2)

41–43

423

119.9 (Phe*), 166.0 (y1), 171.0 (AccQ, b1), 230.0 (a2), 253.0 (y2), 258.0 (b2)

41, 42

437

74.2 (Thr*), 166.0 (y1), 170.9 (AccQ, b1), 243.7 (a2), 267.0 (y2), 271.9 (b2)

41–43

407

120.1 (Phe*), 165.9 (y1), 171.0 (AccQ, b1), 213.8 (a2), 236.9 (y2), 241.9 (b2)

41–43

435

72.1(Val*), 170.9 (AccQ, b1), 242.0 (a2), 264.9 (y2), 270.0 (b2)

42–45

IF, LF¹

449

86.2 (Ile/Leu*), 119.5 (Phe*), 171.0 (AccQ, b1), 256.1 (a2), 279.1 (y2), 283.8 (b2)

439

60.4 (Ser*), 136.1 (Tyr*), 171.0 (AccQ, b1), 182.1 (y1), 229.7 (a2), 257.9 (b2), 269.1 (y2)

423

171.0 (AccQ, b1), 182.0 (y1), 213.8 (a2), 241.9 (b2)

STS

464

59.7 (Ser*), 106.1 (y1), 171.2 (AccQ, b1), 219.9 (a2), 257.6 (b2)

36. 37

GVE²

474

148.0 (y1), 170.8 (AccQ, b1), 227.9 (b2), 303.6 (y3)

36, 37

GIN, GLN²

473

85.8 (Ile/Leu*), 87.3 (Asn*), 133.1 (y1), 171.1 (AccQ, b1), 199.5 (a2), 227.8 (b2), 303.1 (y3)

GIT, GLT²

460

85.9 (Ile/Leu*), 119.8 (y1), 170.9 (AccQ, b1), 199.6 (a2), 228.1 (b2), 290.1 (y3), 340.6 (b3)

ASI, ASL¹

460

44.1 (Ala*), 59.6 (Ser*), 85.7 (Ile/Leu*), 171.0 (AccQ, b1), 241.4 (b2), 289.5 (y3), 329.2 (b3)

AVI, AVL¹

472

44.4 (Ala*), 71.9 (Val*), 170.4 (AccQ, b1), 214.1 (a2), 241.7 (b2), 301.7 (y3), 340.9 (b3)

IAI, IAL, LAI, LAL²

486

86.2 (Ile/Leu*), 170.7 (AccQ, b1), 283.7 (b2), 315.9 (y3), 355.3 (b3)

37–41

SGF

480

60.6 (Ser*), 120.5 (Phe*), 165.9 (y1), 170.4 (AccQ, b1), 222.8 (y2), 258.2 (b2)

37, 38

GTF²

494

73.9 (Thr*), 119.7 (Phe*), 165.8 (y1), 227.7 (b2), 267.1 (y2), 300.7 (a3), 323.7 (y3)

37–40

GAF²

464

120.0 (Phe*), 166.0 (y1), 170.8 (AccQ, b1), 227.9 (b2)

37–42

FQ²

464

84.5 (Gln**), 119.9 (Phe*), 147.4 (y1), 170.7 (AccQ, b1), 289.6 (a2), 318.2 (b2)

AAF²

478

166.0 (y1), 170.8 (AccQ, b1), 241.8 (b2), 307.7 (y3)

GII, GIL, GLI, GLL²

472

86.0 (Ile/Leu*), 131.9 (y1), 170.7 (AccQ, b1), 200.2 (a2), 227.9 (b2), 340.8 (b3)

GGF²

450

120.1 (Phe*), 165.9 (y1), 171.1 (AccQ, b1), 199.7 (a2), 227.8 (b2), 279.8 (y3), 285.0 (b3)

451

88.2 (Asp*), 171.0 (AccQ, b1), 257.7 (a2), 280.7 (y2), 285.9 (b2)

FE²

465

119.9 (Phe*), 147.9 (y1), 171.0 (AccQ, b1), 294.6 (y2), 317.6 (b2)

41, 42

GVF²

492

72.0 (Val*), 119.4 (Phe*), 166.0 (y1), 171.0 (AccQ, b1), 199.8 (a2), 227.9 (b2), 326.5 (b3)

YQ²

480

83.8 (Gln**), 100.7 (Gln*), 136.0 (Tyr*), 147.0 (y1), 170.8 (AccQ, b1), 305.9 (a2), 333.6 (b2)

42–44

NF²

450

86.6 (Asn*), 165.8 (y1), 170.8 (AccQ, b1), 284.9 (b2)

42–44

EF²

465

84.0 (Glu**), 102.1 (Glu*), 120.2 (Phe*), 165.9 (y1), 171.0 (AccQ, b1), 299.8 (b2)

43–45

453

74.0 (Thr*), 136.0 (Tyr*), 271.8 (b2), 282.7 (y2)

44, 45

VY²

451

72.3 (Val*), 170.9 (AccQ, b1), 241.9 (a2), 269.8 (b2), 280.6 (y2)

STE²

506

59.9 (Ser*), 74.1 (Thr*), 147.6 (y1), 170.9 (AccQ, b1), 229.5 (a2), 257.7 (b2), 335.6 (y3)

LTE, ITE²

532

85.8 (Ile/Leu*), 147.7 (y1), 170.9 (AccQ, b1), 255.5 (a2), 361.6 (y3), 385.2 (b3)

TPI, TPL¹

500

69.7 (Pro*), 85.7 (Ile/Leu*), 132.2 (y1), 170.5 (AccQ, b1), 228.8 (y2), 271.8 (b2), 330.1 (y3)

IIQ, ILQ, LIQ, LLQ¹

543

86.1 (Ile/Leu*), 100.8 (Gln*), 147.1 (y1), 171.0 (AccQ, b1), 256.0 (a2), 284.0 (b2), 372.9 (y3), 396.9 (b3)

DVI, DVL¹

516

85.9 (Ile/Leu*), 171.0 (AccQ, b1), 285.6 (b2), 384.9 (b3)

37, 38

IAF, LAF¹

520

86.0 (Ile/Leu*), 165.6 (y1), 171.0 (AccQ, b1), 236.4 (y2), 256.1 (a2), 283.8 (b2), 354.7 (b3)

41–44

NIF, NLF¹

563

86.1 (Ile/Leu*), 119.7 (Phe*), 170.8 (AccQ, b1), 257.1 (a2), 284.9 (b2), 397.8 (b3)

41–44

SIF, SLF¹

536

60.2 (Ser*), 86.1 (Ile/Leu*), 119.9 (Phe*), 165.6 (y1), 170.9 (AccQ, b1), 229.7 (a2), 371.0 (b3)

**Table 2.** Summary of estimated peptide sequences and amino acid derivatives of AccQ-derivatives in the exopeptidase digest of RPH
Peak No.	SEC Fr.	Estimated peptide sequence	Precursor ions (m/z)	Product ions (m/z)
¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides. Supplemental Figure 2 shows the mass chromatograms with peak numbers.
1	36	GP	343	69.7 (Pro*), 116.2 (y1), 171.1 (AccQ, b1), 228.0 (b2)
2	36	GV	345	171.0 (AccQ, b1), 175.0 (y2), 228.0 (b2)
3	34, 35	N-acetyllysine	359	84.0, 127.8, 171.0, 189.1
4	35	SP	373	115.9 (y1), 171.0 (AccQ, b1), 202.6 (y2), 230.2 (a2), 258.2 (b2)
5	35	AP	357	70.0 (Pro*), 116.0 (y1) , 171.0 (AccQ, b1), 187.1 (y2)
6	35	PS	373	70.0 (Pro*), 171.0 (AccQ, b1), 202.9 (y2), 240.0 (a2)
7	35	PP	383	70.0 (Pro*), 115.9 (y1), 171.0 (AccQ, b1), 212.9 (y2), 267.7 (b2)
8	35	PV	385	70.0 (Pro*), 171.0 (AccQ, b1), 215.1 (y2), 240.2 (a2), 268.1 (b2)
9	35	VV	387	72.0 (Val*), 118.3 (y1), 171.0 (AccQ, b1), 216.9 (y2), 241.9 (a2), 270.1 (b2)
10	35, 36	IP, LP¹	399	86.0 (Ile/Leu*), 171.0 (AccQ, b1), 229.0 (y2), 256.2 (a2), 284.0 (b2)
11	36, 37	GQ	374	56.0 (Gln), 84.3 (Gln), 147.3 (y1), 171.0 (AccQ, b1), 200.4 (a2), 228.0 (b2)
12	34, 35	PQ	414	70.0 (Pro*), 171.0 (AccQ, b1), 243.8 (y2)
13	34	pEK	428	84.0 (pyroGlu*), 171.0 (AccQ, b1), 258.2
14	35	QP	414	84.1 (Gln*), 170.9 (AccQ, b1), 244.4 (y2), 299.1 (b2)
15	35, 36	DP	401	115.9 (y1), 171.0 (AccQ, b1), 231.0 (y2), 286.3 (b2)
16	35, 36	EP	415	83.6 (Glu**), 115.9 (y1), 171.0 (AccQ, b1), 271.8 (a2), 299.9 (b2)
17	35–37	DV	403	118.1 (y1), 171.0 (AccQ, b1), 233.2 (y2), 286.2 (b2)
18	35, 36	IV, LV¹	401	86.1 (Ile/Leu*), 170.9 (AccQ, b1), 230.8 (y2), 255.9 (a2)
19	36	DE	433	88.0 (Asp*), 147.7 (y1), 170.9 (AccQ, b1), 262.8 (y2), 285.7 (b2)
20	36, 37	NP	400	115.9 (y1), 171.0 (AccQ, b1), 230.0 (y2), 256.7 (a2), 284.7 (b2)
21	36	DI, DL¹	417	86.1 (Ile/Leu*), 170.9 (AccQ, b1), 247.1 (y2), 286.0 (b2)
22	37–42	FP	433	70.0 (Pro), 116.2 (y1), 119.9 (Phe), 170.9 (AccQ, b1), 290.3 (a2), 318.3 (b2)
23	39–48	YP	449	70.1 (Pro), 115.8 (y1), 136.0 (Tyr), 171.0 (AccQ, b1), 279.2 (y2), 306.2 (a2), 334.0 (b2)
24	39–42	DF	451	120.3 (Phe*), 166.2 (y1), 171.1(AccQ, b1), 285.9 (b2)
25	44–48	TY	453	74.0 (Thr*), 170.9 (AccQ, b1), 272.3 (b2), 282.9 (y2)

Table 2. Summary of estimated peptide sequences and amino acid derivatives of AccQ-derivatives in the exopeptidase digest of RPH

Peak No.

SEC Fr.

Estimated peptide sequence

Precursor ions (m/z)

Product ions (m/z)

¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides. Supplemental Figure 2 shows the mass chromatograms with peak numbers.

343

69.7 (Pro*), 116.2 (y1), 171.1 (AccQ, b1), 228.0 (b2)

345

171.0 (AccQ, b1), 175.0 (y2), 228.0 (b2)

34, 35

N-acetyllysine

359

84.0, 127.8, 171.0, 189.1

373

115.9 (y1), 171.0 (AccQ, b1), 202.6 (y2), 230.2 (a2), 258.2 (b2)

357

70.0 (Pro*), 116.0 (y1) , 171.0 (AccQ, b1), 187.1 (y2)

373

70.0 (Pro*), 171.0 (AccQ, b1), 202.9 (y2), 240.0 (a2)

383

70.0 (Pro*), 115.9 (y1), 171.0 (AccQ, b1), 212.9 (y2), 267.7 (b2)

385

70.0 (Pro*), 171.0 (AccQ, b1), 215.1 (y2), 240.2 (a2), 268.1 (b2)

387

72.0 (Val*), 118.3 (y1), 171.0 (AccQ, b1), 216.9 (y2), 241.9 (a2), 270.1 (b2)

35, 36

IP, LP¹

399

86.0 (Ile/Leu*), 171.0 (AccQ, b1), 229.0 (y2), 256.2 (a2), 284.0 (b2)

36, 37

374

56.0 (Gln**), 84.3 (Gln**), 147.3 (y1), 171.0 (AccQ, b1), 200.4 (a2), 228.0 (b2)

34, 35

414

70.0 (Pro*), 171.0 (AccQ, b1), 243.8 (y2)

pEK

428

84.0 (pyroGlu*), 171.0 (AccQ, b1), 258.2

414

84.1 (Gln*), 170.9 (AccQ, b1), 244.4 (y2), 299.1 (b2)

35, 36

401

115.9 (y1), 171.0 (AccQ, b1), 231.0 (y2), 286.3 (b2)

35, 36

415

83.6 (Glu**), 115.9 (y1), 171.0 (AccQ, b1), 271.8 (a2), 299.9 (b2)

35–37

403

118.1 (y1), 171.0 (AccQ, b1), 233.2 (y2), 286.2 (b2)

35, 36

IV, LV¹

401

86.1 (Ile/Leu*), 170.9 (AccQ, b1), 230.8 (y2), 255.9 (a2)

433

88.0 (Asp*), 147.7 (y1), 170.9 (AccQ, b1), 262.8 (y2), 285.7 (b2)

36, 37

400

115.9 (y1), 171.0 (AccQ, b1), 230.0 (y2), 256.7 (a2), 284.7 (b2)

DI, DL¹

417

86.1 (Ile/Leu*), 170.9 (AccQ, b1), 247.1 (y2), 286.0 (b2)

37–42

433

70.0 (Pro*), 116.2 (y1), 119.9 (Phe*), 170.9 (AccQ, b1), 290.3 (a2), 318.3 (b2)

39–48

449

70.1 (Pro*), 115.8 (y1), 136.0 (Tyr*), 171.0 (AccQ, b1), 279.2 (y2), 306.2 (a2), 334.0 (b2)

39–42

451

120.3 (Phe*), 166.2 (y1), 171.1(AccQ, b1), 285.9 (b2)

44–48

453

74.0 (Thr*), 170.9 (AccQ, b1), 272.3 (b2), 282.9 (y2)

**Table 3.** Summary of estimated sequence of pyroglutamyl peptides in RPH
Peak No.	SEC Fr.	Estimated peptide sequence	Precursor ions (m/z)	Product ions (m/z)
¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides.²The identities of the tentatively assigned peptides were not confirmed, as their ion intensity was low in single ion monitoring mode. Supplemental Figure 3 shows the mass chromatograms with peak numbers.
1	35, 36	pEA	201	44.3 (Ala), 84.1 (pyroGlu), 89.9 (y1), 154.8 (a2), 183.1 (b2)
2	36	pES²	217	60.2 (Ser), 84.3 (pyroGlu), 106.0 (y1), 170.8 (a2), 198.9 (b2)
3	36	pEV	229	72.1 (Val), 84.0 (pyroGlu), 118.0 (y1), 183.0 (a2)
4	36–38	pEI, pEL¹	243	84.2 (pyroGlu), 86.3 (Ile/Leu), 132.4 (y1), 197.2 (a2)
5	35–37	pEQ	258	84.1 (pyroGlu*), 147.1 (y1), 212.3 (a2), 240.0 (b2)
6	35	pEE	259	84.1 (pyroGlu), 101.8 (Glu), 148.1 (y1), 241.2 (b2)
7	41, 42	pEF	277	84.0 (pyroGlu), 120.1 (Phe), 165.9 (y1), 231.1 (a2), 259.3 (b2)
8	44, 45	pEY	293	84.1 (pyroGlu), 136.1 (Tyr), 182.1 (y1), 247.1 (a2)
9	33, 34	pEAQ²	329	44.4 (A), 84.1 (pyroGlu), 100.8 (Q*), 147.0 (y1), 155.2 (a2), 183.3 (b2)
10	33	pEVA²	300	44.4 (Ala), 72.1 (Val), 84.3 (pyroGlu*), 90.2 (y1), 183.0 (a2), 211.0 (b2)
11	33	pELS, pEIS¹	330	60.2 (Ser), 84.2 (pyroGlu), 86.2 (Ile/Leu*), 106.0 (y1), 197.0 (a2), 225.1 (b2), 312.2 (b3)
12	35	pEAE²	330	44.1 (Ala), 83.8 (pyroGlu), 102.3 (Glu*), 147.9 (y1), 154.9 (a2), 182.9 (b2), 219.0 (y2)
13	33, 34	pEQQ²	386	84.1 (pyroGlu), 101.2 (Gln), 147.1 (y1), 257.6 (c2)
14	33, 34	pETQ²	359	84.3 (pyroGlu*), 147.0 (y1), 185.2 (a2), 212.9 (b2), 230.1(c2)
15	33, 34	pEEQ²	387	84.3 (pyroGlu), 101.0 (Gln), 102.3 (Glu*), 147.1 (y1), 212.8 (a2), 241.0 (b2),
16	33, 34	pEQE²	387	84.3 (pyroGlu), 102.3 (Glu), 148.2 (y1), 211.8 (a2), 240.2 (b2)
17	33, 34	pETE²	360	74.3 (Thr), 84.0 (pyroGlu), 102.2 (Glu*), 148.1(y1), 184.7 (a2), 212.8 (b2)
18	33, 34	pEVQ	359	72.4 (Val), 84.3 (pyroGlu), 147.2 (y1), 182.9 (a2), 211.3 (b2), 246.0 (y2)
19	33, 34	pEIQ, pELQ²	371	84.3 (pyroGlu), 86.3 (Ile/Leu), 101.0 (Gln*), 147.1 (y1), 197.1 (a2), 225.2 (b2), 260.1(y2)
20	35	pEQI. pEQL¹	371	84.0 (pyroGlu), 86.1 (Ile/Leu), 132.1 (y1), 212.0 (a2), 240.1 (b2), 324.9 (a3)
21	35–37	pEII, pEIL, pELI, pELL¹	356	84.1 (pyroGlu), 86.2 (Ile/Leu), 132.2 (y1), 197.1 (a2), 225.1 (b2)
22	41	pEVY²	392	72.3 (Val), 84.2 (pyroGlu), 136.0 (Tyr*), 182.1 (y1), 211.3 (b2),
23	32–33	pELSE, pEISE¹	459	60.2 (S), 84.0 (pyroGlu), 86.0 (I/L), 101.9 (E), 148.0 (y1), 197.1 (a2), 225.0 (b2), 235.0 (y2), 283.9 (a3), 312.0 (b3)
24	32–34	pEQII, pEQIL, pEQLI, pEQLL¹	484	83.9 (pyroGlu), 86.1 (Ile/Leu), 101.1 (Q*), 240.1 (b2), 245.0 (y2), 353.1 (b3)
25	37, 38	pEFQ	405	84.0 (pyroGlu), 101.1 (Gln), 120.1 (Phe*), 147.0 (y1), 231.1 (a2)
26	37, 38	pEQF²	405	84.0 (pyroGlu), 119.9 (Phe), 166.0 (y1), 240.1 (b2)

Table 3. Summary of estimated sequence of pyroglutamyl peptides in RPH

Peak No.

SEC Fr.

Estimated peptide sequence

Precursor ions (m/z)

Product ions (m/z)

¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides.²The identities of the tentatively assigned peptides were not confirmed, as their ion intensity was low in single ion monitoring mode. Supplemental Figure 3 shows the mass chromatograms with peak numbers.

35, 36

pEA

201

44.3 (Ala*), 84.1 (pyroGlu*), 89.9 (y1), 154.8 (a2), 183.1 (b2)

pES²

217

60.2 (Ser*), 84.3 (pyroGlu*), 106.0 (y1), 170.8 (a2), 198.9 (b2)

pEV

229

72.1 (Val*), 84.0 (pyroGlu*), 118.0 (y1), 183.0 (a2)

36–38

pEI, pEL¹

243

84.2 (pyroGlu*), 86.3 (Ile/Leu*), 132.4 (y1), 197.2 (a2)

35–37

pEQ

258

84.1 (pyroGlu*), 147.1 (y1), 212.3 (a2), 240.0 (b2)

pEE

259

84.1 (pyroGlu*), 101.8 (Glu*), 148.1 (y1), 241.2 (b2)

41, 42

pEF

277

84.0 (pyroGlu*), 120.1 (Phe*), 165.9 (y1), 231.1 (a2), 259.3 (b2)

44, 45

pEY

293

84.1 (pyroGlu*), 136.1 (Tyr*), 182.1 (y1), 247.1 (a2)

33, 34

pEAQ²

329

44.4 (A*), 84.1 (pyroGlu*), 100.8 (Q*), 147.0 (y1), 155.2 (a2), 183.3 (b2)

pEVA²

300

44.4 (Ala*), 72.1 (Val*), 84.3 (pyroGlu*), 90.2 (y1), 183.0 (a2), 211.0 (b2)

pELS, pEIS¹

330

60.2 (Ser*), 84.2 (pyroGlu*), 86.2 (Ile/Leu*), 106.0 (y1), 197.0 (a2), 225.1 (b2), 312.2 (b3)

pEAE²

330

44.1 (Ala*), 83.8 (pyroGlu*), 102.3 (Glu*), 147.9 (y1), 154.9 (a2), 182.9 (b2), 219.0 (y2)

33, 34

pEQQ²

386

84.1 (pyroGlu*), 101.2 (Gln*), 147.1 (y1), 257.6 (c2)

33, 34

pETQ²

359

84.3 (pyroGlu*), 147.0 (y1), 185.2 (a2), 212.9 (b2), 230.1(c2)

33, 34

pEEQ²

387

84.3 (pyroGlu*), 101.0 (Gln*), 102.3 (Glu*), 147.1 (y1), 212.8 (a2), 241.0 (b2),

33, 34

pEQE²

387

84.3 (pyroGlu*), 102.3 (Glu*), 148.2 (y1), 211.8 (a2), 240.2 (b2)

33, 34

pETE²

360

74.3 (Thr*), 84.0 (pyroGlu*), 102.2 (Glu*), 148.1(y1), 184.7 (a2), 212.8 (b2)

33, 34

pEVQ

359

72.4 (Val*), 84.3 (pyroGlu*), 147.2 (y1), 182.9 (a2), 211.3 (b2), 246.0 (y2)

33, 34

pEIQ, pELQ²

371

84.3 (pyroGlu*), 86.3 (Ile/Leu*), 101.0 (Gln*), 147.1 (y1), 197.1 (a2), 225.2 (b2), 260.1(y2)

pEQI. pEQL¹

371

84.0 (pyroGlu*), 86.1 (Ile/Leu*), 132.1 (y1), 212.0 (a2), 240.1 (b2), 324.9 (a3)

35–37

pEII, pEIL, pELI, pELL¹

356

84.1 (pyroGlu*), 86.2 (Ile/Leu*), 132.2 (y1), 197.1 (a2), 225.1 (b2)

pEVY²

392

72.3 (Val*), 84.2 (pyroGlu*), 136.0 (Tyr*), 182.1 (y1), 211.3 (b2),

32–33

pELSE, pEISE¹

459

60.2 (S*), 84.0 (pyroGlu*), 86.0 (I/L*), 101.9 (E*), 148.0 (y1), 197.1 (a2), 225.0 (b2), 235.0 (y2), 283.9 (a3), 312.0 (b3)

32–34

pEQII, pEQIL, pEQLI, pEQLL¹

484

83.9 (pyroGlu*), 86.1 (Ile/Leu*), 101.1 (Q*), 240.1 (b2), 245.0 (y2), 353.1 (b3)

37, 38

pEFQ

405

84.0 (pyroGlu*), 101.1 (Gln*), 120.1 (Phe*), 147.0 (y1), 231.1 (a2)

37, 38

pEQF²

405

84.0 (pyroGlu*), 119.9 (Phe*), 166.0 (y1), 240.1 (b2)

**Table 4.** Summary of estimated sequence of pyroglutamyl peptides and related compound in the exopeptidase digest of RPH
Peak No.	SEC Fr.	Estimated peptide sequence	Precursor ions (m/z)	Product ions (m/z)
¹Presence of both peptides consisting of Leu and Ile were confirmed by comparison with synthetic peptides. ²The identities of the tentatively assigned peptides were not confirmed, as their ion intensity was low in single ion monitoring mode. Supplemental Figure 4 shows the mass chromatograms with peak numbers.
1	36	pES²	217	56.2 (pyroGlu*), 60.2 (Ser), 84.1(pyroGlu*), 106.0 (y1), 199.3 (b2)
2	36, 37	pEV	229	72.0 (Val), 84.0 (pyroGlu), 118.0 (y1), 183.0 (a2)
3		two pyroGlu	259
4	35–37	pEQ	258	56.1 (pyroGlu, Gln), 84.0 (pyroGlu, Gln*), 146.8 (y1)
5	36, 37	pEE	259	55.9 (pyroGlu*), 84.0 (pyroGlu), 102.0 (Glu*), 148.0 (y2), 241.0 (b2)
6	37, 38	pEI, pEIL¹	243	56.0 (pyroGlu*), 84.0 (pyroGlu), 86.0 (Ile/Leu*), 128.9 (c1), 132.0 (y1), 197.0 (a2)
7	42–46	pEY	293	84.0 (pyroGlu), 136.0 (Tyr), 182.0 (y1), 247.0 (a2)
8	41–46	pEF	277	56.0 (pyroGlu*), 84.0 (pyroGlu), 120.0 (Phe*), 166.0 (y1), 231.0 (a2)
9	34	pEQP	355	70.2 (Pro), 84.0 (pyroGlu), 100.8 (Gln*), 115.8 (y1), 239.9 (b2), 338.4 (b3)
10	34	pENS²	331	60.0 (Ser), 84.0 (pyroGlu), 106.0 (y1), 198.0 (a2), 313.0 (b3)
11	35	pEDV²	344	72.1 (Val), 83.9 (pyroGlu), 118.0 (y1), 227.0 (b2), 233.0 (y2), 298.3 (a3)

Table 4. Summary of estimated sequence of pyroglutamyl peptides and related compound in the exopeptidase digest of RPH

Peak No.

SEC Fr.

Estimated peptide sequence

Precursor ions (m/z)

Product ions (m/z)

pES²

217

56.2 (pyroGlu**), 60.2 (Ser*), 84.1(pyroGlu*), 106.0 (y1), 199.3 (b2)

36, 37

pEV

229

72.0 (Val*), 84.0 (pyroGlu*), 118.0 (y1), 183.0 (a2)

two pyroGlu

259

35–37

pEQ

258

56.1 (pyroGlu**, Gln**), 84.0 (pyroGlu*, Gln**), 146.8 (y1)

36, 37

pEE

259

55.9 (pyroGlu**), 84.0 (pyroGlu*), 102.0 (Glu*), 148.0 (y2), 241.0 (b2)

37, 38

pEI, pEIL¹

243

56.0 (pyroGlu**), 84.0 (pyroGlu*), 86.0 (Ile/Leu*), 128.9 (c1), 132.0 (y1), 197.0 (a2)

42–46

pEY

293

84.0 (pyroGlu*), 136.0 (Tyr*), 182.0 (y1), 247.0 (a2)

41–46

pEF

277

56.0 (pyroGlu**), 84.0 (pyroGlu*), 120.0 (Phe*), 166.0 (y1), 231.0 (a2)

pEQP

355

70.2 (Pro*), 84.0 (pyroGlu*), 100.8 (Gln*), 115.8 (y1), 239.9 (b2), 338.4 (b3)

pENS²

331

60.0 (Ser*), 84.0 (pyroGlu*), 106.0 (y1), 198.0 (a2), 313.0 (b3)

pEDV²

344

72.1 (Val*), 83.9 (pyroGlu*), 118.0 (y1), 227.0 (b2), 233.0 (y2), 298.3 (a3)