Characterisation of TNF-α-related peptides by high-performance liquid chromatography—mass spectrometry and high-performance liquid chromatography—tandem mass spectrometry

Abstract

Ionspray triple quadrupole mass spectrometry and high-performance liquid chromatography were used to investigate the products from the solid phase synthesis of (H)—Leu—Thr—Glu—Asn—(OH), a TNF-α active-site probe. The target sequence was assembled using tert.-butoxycarbonyl (Boc) chemistry in stepwise fashion from the C-terminal on an Boc—Asn—OCH 2—Pamcopoly) (styrene—divinylbenzene) resin [Pam = 4-(carboxamidomethyl)benzyl ester]. The crude product was deprotected and cleaved from the resin by HF- p-cresol treatment for 1 h at 0°C. HPLC analysis at 214 nm indicated two late-eluting major products and an early-eluting product. Preparative HPLC demonstrated that the early-eluting product contained ca. 80% of the expected recovered sample mass. Each component was then directly analysed spectrometry and tandem mass spectrometry. The early eluting peak was confirmed as the LTEN sequence. Synthesis of the same sequence using 9-fluorenyl methoxycarbonly (Fmoc) chemistry gave an identical product and comfirmed the above analysis. The most significant by-product was derived from arylation of the glutamyl group by the quencher p-cresol. The likely origins of the by-products are discussed.