Ultrasonic-assisted extraction and digestion of proteins from solid biopsies followed by peptide sequential extraction hyphenated to MALDI-based profiling holds the promise of distinguishing renal oncocytoma from chromophobe renal cell carcinoma

• Published in: Talanta (Oxford) 2020-01, Vol.206, p.120180-120180, Article 120180
• Main Authors: Jorge, Susana, Pereira, Kevin, López-Fernández, Hugo, LaFramboise, William, Dhir, Rajiv, Fernández-Lodeiro, Javier, Lodeiro, Carlos, Santos, Hugo M., Capelo-Martínez, Jose L.
• Format: Article
• Language: English
• Subjects: Acetonitriles - chemistry; Adenoma, Oxyphilic - diagnosis; Adult; Aged; Aged, 80 and over; Animals; Biomarkers, Tumor - analysis; Biomarkers, Tumor - chemistry; Biomarkers, Tumor - isolation & purification; Biopsy; Carcinoma, Renal Cell - diagnosis; Chromophobe renal cell carcinoma; Diagnosis, Differential; Enzymes, Immobilized - chemistry; Female; Humans; Kidney - chemistry; Kidney - pathology; Kidney Neoplasms - diagnosis; Magnetite Nanoparticles - chemistry; MALDI; Male; Mice; Middle Aged; Peptide Fragments - analysis; Profiling ESI; Proteins - analysis; Proteins - chemistry; Proteins - isolation & purification; Proteomics - methods; Renal cancer; Renal oncocytoma; Sequential extraction; Solid Phase Extraction - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Trypsin - chemistry; Ultrasonic Waves
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2019.120180

A novel analytical approach is proposed to discriminate between solid biopsies of chromophobe renal cell carcinoma (chRCC) and renal oncocytoma (RO). The method comprises the following steps: (i) ultrasonic extraction of proteins from solid biopsies, (ii) protein depletion with acetonitrile, (iii) ultrasonic assisted in-solution digestion using magnetic nanoparticle with immobilized trypsin, (iv) C18 tip-based preconcentration of peptides, (v) sequential extraction of the peptides with ACN, (vi) MALDI-snapshot of the extracts and (vii) investigation of the extract containing the most discriminating features using high resolution mass spectrometry. With this approach we have been able to differentially cluster renal oncocytoma and chromophobe renal cell carcinoma and identified 18 proteins specific to chromophobe and seven unique to renal oncocytoma. Chromophobes express proteins associated with ATP function (ATP5I & 5E; VATE1 & G2; ADT2), glycolysis (PGK1) and neuromedin whilst oncocytomas express ATP5H, ATPA, DEPD7 and TRIPB thyroid receptor interacting protein. [Display omitted] •A new fast, simple and low-cost method for analysis of solid biopsies is presented.•Two renal tumors, chromophobe renal cell carcinoma and renal oncocytoma, were discriminated.•Acetonitrile-based depletion was used to reduce the complexity of proteomes.•Peptide sequential elution was applied to fractionate the proteome.•The 60% (v/v) acetonitrile elution fraction was the most informative for tumor discrimination.