A novel strategy for the discrimination of gelatinous Chinese medicines based on enzymatic digestion followed by nano-flow liquid chromatography in tandem with orbitrap mass spectrum detection

Figures & data

Table 1 Gelatinous Chinese medicines in the Chinese Pharmacopoeia³

Latin name	Common names	Animal origins	Medicinal parts	Dosage (g/day)
Asini Corii Colla	Donkey-hide glue, E Jiao	Equus asinus L.	Skin	3–9
Cervi Cornus Colla	Deer-horn glue, Lu Jiao Jiao	Cervus elaphus Linnaeus, Cervus nippon Temminck	Ossified antlers or horn base (naturally detach after cutting pilose non-ossified antlers from deer)	3–6
Testudinis Carapacis ET Plastri Colla	Tortoise-shell glue, Gui Jia Jiao	Chinemys reevesii (Gray)	Carapace and plastron	3–9

Figure 1 Quality control scheme of GCMs in the ChP.

Abbreviations: ACC, Asini Corii Colla; CCC, Cervi Cornus Colla; ChP, Chinese Pharmacopoeia; GCM, gelatinous Chinese medicine; TCPC, Testudinis Carapacis ET Plastri Colla; TLC, thin layer chromatography.

Figure 2 Novel strategy proposed for the discrimination of GCMs.

Abbreviations: GCM, gelatinous Chinese medicine; MSⁿ, multi-stage mass spectrometry; NanoLC-orbitrap MSD, nano-flow liquid chromatography in tandem with orbitrap mass spectrum detection.

Table 2 Calibration curve and protein content

Assays	Regression equations	r^2	Linearity ranges	Protein content (w/w)
				ACC	CCC	TCPC
BCA	Y=0.0010X +0.0651	0.9993	62.50–2,000 μg/mL	44.2%	40.7%	36.4%
Bradford	Y=0.0056X +0.0729	0.9995	20.00–100.0 μg/mL	19.3%	15.9%	14.4%

Abbreviations: ACC, Asini Corii Colla; BCA, bicinchoninic acid; CCC, Cervi Cornus Colla; TCPC, Testudinis Carapacis ET Plastri Colla; r², correlation coefficient; w/w: weight/weight.

Figure 3 SDS–PAGE analysis of gelatinous Chinese medicines and their digests (1.0 mm, 5% stacking gel, 10% resolving gel, silver staining).

Notes: Lane 1: ladder. Lane 2: collagenase after incubation. Lane 3: ACC. Lane 4: ACC after in-solution digestion by collagenase. Lane 5: CCC. Lane 6: CCC after in solution digestion by collagenase. Lane 7: TCPC. Lane 8: TCPC after in solution digestion by collagenase. Lane 9: gelatin after in solution digestion by collagenase. Lane 10: gelatin.

Abbreviations: ACC, Asini Corii Colla; CCC, Cervi Cornus Colla; Mw, molecular weight; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCPC, Testudinis Carapacis ET Plastri Colla.

Figure 4 NanoLC-orbitrap MSD TIC of gelatinous Chinese medicines’ digests.

Notes: NanoLC-orbitrap MSD TIC of (A) ACC (B/N: 110916), (B) CCC (B/N: 20130922), and (C) TCPC (B/N: 20131018).

Abbreviations: ACC, Asini Corii Colla; CCC, Cervi Cornus Colla; NanoLC-orbitrap MSD, nano-flow liquid chromatography in tandem with orbitrap mass spectrum detection; TCPC, Testudinis Carapacis ET Plastri Colla; TIC, total ion chromatogram; B/N, batch number; NL, normalized Level; RT, retention time.

Table 3 List of potential biomarkers for individual GCMs

Codes	Potential biomarkers	GCMs	NanoLC		Orbitrap MSD		PEAKS
			tR (min)	Charges	Precursor MH^2+	Precursor mass	Mass error	ALC
A1	GFPGAVGAKGEGGGAP	ACC	18.07	2+	664.8349	1,327.652	2.4 ppm	95%
A2	FGSGLDKADGGAGAP	ACC	26.13	2+	660.3151	1,318.615	0.3 ppm	94%
A3	LAGAPGFRAGP	ACC	16.87	2+	523.2746	1,044.535	−0.5 ppm	94%
A4	FSGLDGSVAGGAGAP	ACC	19.72	2+	631.8045	1,261.594	0.6 ppm	93%
A5	QTGFPGAAGGVVGPP	ACC	17.53	2+	680.3351	1,358.647	6.8 ppm	93%
C1	GGPGSVFGGPGADGVAGKP	CCC	14.26	3+	539.2655	1,614.764	6.8 ppm	94%
C2	QPGTPGPEGLATQGP	CCC	17.17	2+	719.8500	1,437.674	8.2 ppm	92%
C3	VAGEPAAADGLGPGGP	CCC	15.41	2+	684.3293	1,366.637	5.5 ppm	91%
C4	FSGLDGASVGGAGAP	CCC	22.90	2+	631.8044	1,261.594	0.5 ppm	90%
T1	QTGFPGAAGRVGPP	TCPC	17.93	2+	680.3376	1,358.658	2.2 ppm	95%
T2	QPGFPGARGPAGGAP	TCPC	15.99	2+	692.8358	1,383.653	2.8 ppm	95%
T3	QVGPAGLNAVQGSGAGAP	TCPC	16.00	2+	783.9045	1,565.78	9.3 ppm	93%
T4	LAGAPGFRQP	TCPC	17.28	2+	523.2755	1,044.535	1.3 ppm	92%
T5	FSGLDGKDAGAGGAP	TCPC	24.64	2+	660.3176	1,318.615	4.1 ppm	92%

Abbreviations: ACC, Asini Corii Colla; ALC, average local confidence; CCC, Cervi Cornus Colla; GCM, Gelatinous Chinese medicine; MSD, mass spectrum detection; NanoLC, nano-flow liquid chromatography; TCPC, Testudinis Carapacis ET Plastri Colla; t_R, retention time.

Table 4 List of sequences producing significant alignments by PSI-BLAST against nrdb

GCM	Potentialbiomarker	Description	Max score	Total score	Query cover	Sequenceidentity	Accessions
ACC	GFPGAVGA KGEGGGAP	Predicted: collagen alpha-1(I) chain (Equus caballus)	37.1	79.5	100%	82%	XP_005597538.1
		Predicted: collagen alpha-1(I) chain (Equus przewalskii)	37.1	97.6	100%	82%	XP_008516208.1
		Collagen alpha-1 type I chain (Equus asinus L)	37.1	120	100%	82%	ACM24774.1

Notes: Max score is the score of single best aligned sequence. Total score is sum of all aligned sequence scores. Query cover is what percent of query sequence is aligned.

Abbreviations: ACC, Asini Corii Colla; GCM, Gelatinous Chinese medicine; nrdb, non-redundant protein sequences databases; PSI-BLAST, Position-Specific Iterated Basic Local Alignment Search Tool.

Chinese Pharmacopoeia CommitteeChinese PharmacopoeiaI.2010BeijingChinese Medical Science and Technology Press2010

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