Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

Abstract

Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14-3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions.

Keywords: 14-3-3ε; FASN; FFPE; mass spectrometry; melanoma; proteomics.

Figure 1.

STRING analysis of metabolic and PI3K-Akt signalling pathways. STRING analysis, generating functional protein association networks, displays 64 proteins discovered to be upregulated in invasive melanoma compared with in situ melanoma, as listed in Table II. Highlighted in red are nine proteins that participate in metabolism (TPI1, PRDX6, ALDOA, ACSL1, MDH2, GAPDH, PGK1, PKM and FASN) and eight proteins that participate in the PI3K-Akt signalling pathway (YWHAE, YWHAQ, HSP90B1, YWHAB, YWHAZ, HSP90AB1, COL6A2 and COL6A1). PI3K, phosphoinositide 3-kinase; TPI1, triosephosphate isomerase; PRDX6, peroxiredoxin-6; ALDOA, fructose-bisphosphate aldolase A; ACSL1, acyl-CoA synthetase long-chain family member 1; MDH2, malate dehydrogenase, mitochondrial; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase 1; PKM, pyruvate kinase isozymes M1/M2; FASN, fatty acid synthase; YWHAE, 14-3-3ε; YWHAQ, 14-3-3ζ; HSP90B1, endoplasmin; YWHAB, 14-3-3α/β; YWHAZ, 14-3-3ζ/δ; HSP90AB1, heat shock protein HSP 90-β; COL6A2, collagen α-2(VI) chain; COL6A1, collagen α-1(VI) chain.

Figure 2.

IHC analysis of 14-3-3ε and FASN in primary and metastatic melanoma. (A) Representative photomicrograph showing decreased 14-3-3ε immunoreactivity in primary melanoma tumour tissue compared with metastatic melanoma tissue. (B) Representative photomicrograph showing increased 14-3-3ε immunoreactivity in metastatic melanoma tumour tissue compared with primary melanoma tissue. (C) Representative photomicrograph showing decreased 14-3-3ε immunoreactivity in primary melanoma tumour tissue compared with metastatic melanoma tissue. (D) Representative photomicrograph showing increased 14-3-3ε immunoreactivity in metastatic melanoma tumour tissue compared with primary melanoma tissue. (E) Positive control. Strong FASN immunoreactivity is observed in invasive ductal breast carcinoma. (F) Representative photomicrograph showing increased FASN immunoreactivity in metastatic melanoma tumour tissue compared with primary melanoma tissue. (G) Representative photomicrograph showing decreased FASN immunoreactivity in primary melanoma tumour tissue. (H) Representative photomicrograph showing increased FASN immunoreactivity in metastatic melanoma tumour tissues compared with primary melanoma tissue. Magnification, ×400. Scale bar=200 µm. FASN, fatty acid synthase.