SERUM ÖRNEKLERİNDE ALFA-2-MAKROGLUBULİN TAYİNİ

Amaç: Proteomik, klinik çalışmalarda yaygın olarak kullanılan en hızlı büyüyen omiklerden biridir. Proteomik, tek bir hücreden başlayarak karmaşık biyolojik örneklere kadar geniş bir örnek yelpazesinde kalitatif ve kantitatif protein analizini içerir. Protein bazlı biyobelirteç çalışmaları, metabolik hastalıklar, kanser ve nöropsikiyatrik hastalıklar dahil olmak üzere birçok hastalığa hem tanısal hem de prognostik amaçlarla uygulanmıştır. Alfa-2-makroglubulin (A2MG), kan pıhtılaşması, protein bağlanması ve proteaz inhibisyonu dahil olmak üzere çeşitli biyolojik süreçlerde yer alan, klinik önemi olan ve salgılanan bir proteindir. A2MG analizi için mevcut yöntemler, belirli bir protein birimi veya benzersiz bir peptit yoluyla immün spesifik bağlanmaya odaklandıklarından sınırlıdır. Tek bir protein farklı formlarda (kompleksler, modifikasyonlar, vb.) olabileceğinden ve biyolojik aktivite yapıya özel olduğundan, kapsamlı bir analiz gereklidir. Bu çalışmada kapsamlı A2MG analizi için yeni bir Kütle Spektrometresi (MS) tabanlı yöntem geliştirildi. Gereç ve Yöntem: Bu çalışmada, kapsamlı A2MG analizi için yeni bir Kütle Spektrometresi (MS) tabanlı yöntem geliştirilmiştir. Analitik yöntem geliştirme referans insan serumu ve A2MG protein standardı ile yapılmıştır. Proteolitik protein sindirimi için tripsin kullanılmış ve sindirimden önce ve sonra proteinin denatürasyonu Dairesel-Dikroizm (CD) spektroskopisi kullanılarak test edilmiştir. Hedefli MS yöntemi, serumda A2MG için 12 benzersiz peptidi izlemek için geliştirilmiştir. Sonuç ve Tartışma: Bu çalışmada, biyolojik farklılıkları gözlemlemek için geliştirilen tek bir protein için çoklu peptitlerin ölçülmesi ile sağlam ve güvenilir serumda A2MG analizi geliştirilmiştir. Yöntem, diğer proteinlere de kolayca uygulanabilir. Hedeflenen MS konsepti, daha sonra klinik laboratuvarlara kolayca aktarılabilen ideal bir niceleme ve doğrulama platformu sağlayacaktır.

Anahtar Kelimeler:

Alpha-2-Macroglobulin, Serum, Hedefli proteomik

DETERMINATION OF ALPHA-2-MACROGLUBULIN IN SERUM SAMPLES

Objective: Proteomics is one of the fastest growing omics that has been extensively used in clinical studies. Proteomics involves qualitative and quantitative protein analysis in a wide range of samples starting from a single cell to complex biological samples. Protein-based biomarker studies have been applied to many diseases including metabolic diseases, cancer and neuropsychiatric diseases for both diagnostic and prognostic purposes. Alpha-2-macroglubulin (A2MG) is a clinically relevant secreted protein involving in various biological processes including blood coagulation, protein binding and protease inhibition. Current methods for A2MG analysis are limited, as they focus on either immune-specific binding through a certain protein unit or a unique peptide. As a single protein could be in different forms (complexes, modifications, etc) and the biological activity is structure specific, an extensive analysis is necessary. Here a new Mass-Spectrometry (MS) based method was developed for comprehensive A2MG analysis. Material and Method: A reference human serum and A2MG protein standard were used for method development. Proteolytic protein digestion was performed using trypsin and Circular-Dichroism (CD) spectroscopy was used to ensure protein unfolding and denaturation prior to digestion. Targeted MS method was developed to monitor 12 unique peptides for A2MG in serum. Result and Discussion: Monitoring multiple peptides for a single protein enabled to observe biological differences offer a robust and reliable A2MG analysis in serum. The method can also easily be implemented to other proteins. The concept of targeted-MS provides an ideal quantification and validation platform which then can be easily transferred to clinical laboratories.

Keywords:

Alpha-2-Macroglobulin, Serum, Targeted proteomics,

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