Mass-spectrometric mapping of VDAC (voltage dependent anion channel) from mouse brain membranes with high sequence coverage

Abstract

VDACs (voltage dependent anion channels) are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through mitochondrial membranes. The functional role of VDAC has been investigated in many studies, and different functions of VDAC have been shown. Posttranslational modifications of VDAC are significant for its regulation. The aim of our research was to develop approach for characterization of primary structure and posttranslational modifications of VDACs and other membrane proteins. Mouse brain membranes were isolated from mouse brains by differential centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse brain membranes has been identified almost completely (95 %, 258 of 283 amino acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures after proteolytic degradation with trypsin. Sequence of each found peptide of VDAC has been analysed and confirmed according to accurate mass, isotopic distribution and MS/MS tandem analysis. Posttranslational modifications of VDAC’speptides have been shown. High sequence coverage of VDAC has been obtained, including 11 transmembranes domains. Extensive sequence coverage has been also detected for some other proteins at 30-34 kDa. A repressor of estrogen receptor activity has been identified with 76 % coverage, malate dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have been sequenced with 60 % and 65 % coverage, respectively. These results demonstrated that mass spectrometric mapping is reliable and sensitive approach for characterization of primary structure membrane proteins and identification of their posttranslational modifications. 

Keywords

• VDAC,primary structure

Yüksek dizi kapsama alanı ile fare beyin zarlarından VDAC (voltaj bağımlı anyon kanalı) kütle spektrometrik haritalama.

Öz

VDAK'lar (voltaj bağımlı anyon kanalları) spesifik olmayan difüzyon gözenekleri veya mitokondriyal membran aracılı substratların taşınması için özel sistemler olarak hizmet veren entegre membran proteinleridir. VDAK'ın fonksiyonel rolü birçok çalışmada araştırılmış ve VDAK'ın farklı işlevleri gösterilmiştir. VDAK'ın post-translasyon modifikasyonları regülasyonu için önemlidir. VDAK'ın post-translasyon modifikasyonları regülasyonu için önemlidir. Araştırmamızın amacı, VDAK'ların post-translasyonel ve diğer zar proteinlerinin modifikasyonları ve primer yapının karakterizasyonu için yaklaşım geliştirmektir. Fare beyin zarları diferansiyel santrifüjleme ile fare beyninden izole edildi. Fare beyin zarlarından gelen mitokondriyal izoform VDAK'ın primer yapısı tripsin ile proteolitik yıkımdan sonra peptit karışımlarının LTQ-FTMS haritalama ve SDS-PAGE 'nin kombinasyonu ile  neredeyse tamamı (% 95, 283 amino asitin 258'i) tanımlanmıştır. VDAK’ın bulunan her bir peptid dizisi doğru kütle, izotopik dağılım ve MS / MS tandem analizine göre analiz edilmiş ve doğrulanmıştır. VDAK’ın peptitlerinin post-translasyon modifikasyonları gösterilmiştir. VDAK'ın yüksek dizi içeriği 11 transmembran alanı dahil olmak üzere elde edilmiştir.  30-34 kDa'da diğer bazı proteinler için geniş çaplı sekans içeriği de tespit edilmiştir. Sırasıyla; östrojen reseptorü baskılayıcısı aktivitesinin içeriğinin %76’sı, malat dehidrogenazın %55’i, sintaksin 1A ve sintaksin 1B2’nin %60 ve %65’i belirlenmiştir. Bu sonuçlar, kütle spektrometrik haritalamanın ana yapı membran proteinlerinin karakterizasyonu ve bunların post-translasyonel modifikasyonlarının tanımlanması için güvenilir ve duyarlı bir yaklaşım olduğunu göstermiştir.

Anahtar Kelimeler

• VDAC,birincil yapı

Kaynakça

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