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

Yıl 2018, , 225 - 248, 29.12.2018

Natalia Akentıeva

https://doi.org/10.26453/otjhs.458683

Öz

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. 

Anahtar Kelimeler

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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