<abstract xmlns="http://www.w3.org/1999/xhtml">

<sec><h3>Introduction</h3>
<p>There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in individuals with a light skin/red hair phenotype. However, nothing is known about possible correlation between the type and/or clinical stage of melanoma and the content of pheomelanin in the tumor tissue. We believe that archival formalin-fixed and paraffin-embedded (FFPE) melanoma tissues could be a good source of melanin pigment for future large-scale research on that issue.</p>
</sec>
<sec><h3>Aim</h3>
<p>The aim of this work was to develop a method for isolation and purification of melanin from FFPE samples of human melanoma. To test the suitability of the isolation protocol for planned structural studies, the obtained melanin was analyzed for pheomelanin content by the method based on pyrolysis (Py) coupled with gas chromatography and tandem mass spectrometry (GC/MS/MS).</p>
</sec>
<sec><h3>Material and methods</h3>
<p>For melanin isolation, microtome sections of FFPE tissue of primary lesion and lymph node metastases were subjected to a multistep procedure of paraffin removal, tissue rehydration, homogenization, and digestion with the set of proteolytic enzymes. The pigment samples were then pyrolyzed at 500 °C, and the GC-separated thermal degradation products were identified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode.</p>
</sec>
<sec><h3>Results</h3>
<p>GC/MS/MS analysis of the pyrolysis products revealed the presence of pheomelanin markers, which allowed quantitation of a pheomelanin component of each of the isolated pigments. Melanin from the FFPE primary melanoma was found to contain 6.6%, and the pigment from FFPE metastatic lymph node 7.5% of pheomelanin.</p>
</sec>
<sec><h3>Conclusions</h3>
<p>The developed protocol allows for the isolation of melanin from FFPE melanoma specimens. The pigment can be successfully studied for pheomelanin content by Py-GC/MS/MS method. The results of our study indicate that archival FFPE tumor tissues can be used as a good source of melanin for future structural studies aimed at shedding more light on the role of pheomelanin in the pathomechanism of cutaneous melanoma.</p>
</sec>
</abstract>

Introduction
There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in individuals with a light skin/red hair phenotype. However, nothing is known about possible correlation between the type and/or clinical stage of melanoma and the content of pheomelanin in the tumor tissue. We believe that archival formalin-fixed and paraffin-embedded (FFPE) melanoma tissues could be a good source of melanin pigment for future large-scale research on that issue.

Aim
The aim of this work was to develop a method for isolation and purification of melanin from FFPE samples of human melanoma. To test the suitability of the isolation protocol for planned structural studies, the obtained melanin was analyzed for pheomelanin content by the method based on pyrolysis (Py) coupled with gas chromatography and tandem mass spectrometry (GC/MS/MS).

Material and methods
For melanin isolation, microtome sections of FFPE tissue of primary lesion and lymph node metastases were subjected to a multistep procedure of paraffin removal, tissue rehydration, homogenization, and digestion with the set of proteolytic enzymes. The pigment samples were then pyrolyzed at 500 °C, and the GC-separated thermal degradation products were identified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode.

Results
GC/MS/MS analysis of the pyrolysis products revealed the presence of pheomelanin markers, which allowed quantitation of a pheomelanin component of each of the isolated pigments. Melanin from the FFPE primary melanoma was found to contain 6.6%, and the pigment from FFPE metastatic lymph node 7.5% of pheomelanin.

Conclusions
The developed protocol allows for the isolation of melanin from FFPE melanoma specimens. The pigment can be successfully studied for pheomelanin content by Py-GC/MS/MS method. The results of our study indicate that archival FFPE tumor tissues can be used as a good source of melanin for future structural studies aimed at shedding more light on the role of pheomelanin in the pathomechanism of cutaneous melanoma.

Development of a method for isolation of melanin from archival FFPE tissues of human melanoma for structural studies by pyrolysis-gas chromatography-tandem mass spectrometry

Department of Instrumental Analysis, Faculty of Pharmaceutical Sciences in Sosnowiec

Medical University of Silesia in Katowice

Postępy Higieny i Medycyny Doświadczalnej

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Introduction
There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in...

Time segment	Retention time [min]	Compound (Type^*)	MRM transition		Collision energy [V]

			Precursor Ion [m/z]	Product Ion [m/z]

1	9.7	Thiazole (P1)	85	58	15
			58	57	12
	11.1	Toluene (NS1)	92	91	15
			91	65	16

2	16.2	Styrene (NS2)	104	77	27
			104	78	15
			103	77	14

3	19.2	Phenol (NS3)	94	65	26
			94	66	16
	22.1	Methylphenol (NS4)	108	107	16
			107	77	16

4	24.9	1,2-Benzenediol (NS5)	110	63	30
			110	64	20
			110	92	15
	25.8	Benzothiazole (P2)	135	108	18
			108	82	12

5	26.9	Indole (NS6)	117	89	30
			117	90	16
			90	63	27

6	28.2	4-Hydroxybenzothiazole (P3)	151	96	23
			151	123	12
			123	96	10
	28.5	Methylindole (NS7)	130	77	27
			130	103	14
			103	77	10

7	29.3 – 29.9	2,3-Dihydro-5H-1,4-benzothiazin-5-one (P4) and its isomers (P4′ and P4″)	165	110	18
			165	136	22
			136	109	16

8	30.7 – 30.9	Methyl-2,3-dihydro-5H-1,4-benzothiazin-5-one (P5, P5′)	179	110	15
			179	150	20
			178	109	20
	31.2	4-Hydroxy-6-ethylbenzothiazole (P6)	179	164	15
			164	109	20
			164	136	15

Development of a method for isolation of melanin from archival FFPE tissues of human melanoma for structural studies by pyrolysis-gas chromatography-tandem mass spectrometry

Article Category: Original Study

Publicado en línea: 08 abr 2022

Páginas: 122 - 127

Recibido: 30 mar 2021

Aceptado: 05 ago 2021

DOI: https://doi.org/10.2478/ahem-2022-0006

Palabras clave
FFPE, melanoma, pheomelanin, pyrolysis, GC/MS/MS

© 2022 Sławomir Kurkiewicz, et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Multiple reaction monitoring (MRM) settings for the analysis of melanin pyrolysate by GC/MS/MS

Development of a method for isolation of melanin from archival FFPE tissues of human melanoma for structural studies by pyrolysis-gas chromatography-tandem mass spectrometry

Article Category: Original Study

Publicado en línea: 08 abr 2022

Páginas: 122 - 127

Recibido: 30 mar 2021

Aceptado: 05 ago 2021

DOI: https://doi.org/10.2478/ahem-2022-0006

Palabras claveFFPE, melanoma, pheomelanin, pyrolysis, GC/MS/MS

© 2022 Sławomir Kurkiewicz, et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Multiple reaction monitoring (MRM) settings for the analysis of melanin pyrolysate by GC/MS/MS

Palabras clave
FFPE, melanoma, pheomelanin, pyrolysis, GC/MS/MS