Unearthing Bulgakov's trace proteome from the Master i Margarita manuscript

Highlights

• A new capturing film containing strong cation (SCX) and strong anion (SAX) exchangers is reported.

• Three biomarkers of renal diseases were identified on the original manuscript.

• Twenty nine additional proteins from saliva and skin were also detected.

• Two bacterial proteins on the margins of the foils were identified.

Abstract

Ten pages, selected from a total of 127, of the last manuscript of Master i Margarita, written by Bulgakov in the last four years of his life, have been analysed in order to harvest and identify any trace proteome left on the margin by the novelist, in the hope of finding biomarkers of his fatal nephrotic syndrome. To that aim, we prepared a special ethyl-vinyl acetate film as binder of ground AG 501 Bio-Rad mix-bed strong cation/strong anion exchange resins for adsorbing any protein left on the margins of the pages via saliva and/or sweat. After eluting, digesting and interrogating the peptides by LC-MS/MS, we could identify three proteins, periostin, N-acetyl-β-glucosaminidase and nephrin, reported as biomarkers of renal pathologies. Additionally a further 29 unique gene products, of saliva and skin origin, have been identified, together with two bacterial proteins. The novel method here reported could be safely applied to any other research on manuscripts stored in public libraries and repositories of the World Cultural Heritage.

Significance

The present manuscript aims at finding proteomics traces in a 75-year old manuscript in order to confirm the health state of the author. In the case of Bulgakov it was known that he died of renal disease, possibly leaving traces and/or biomarkers of this pathology on the margins of the pages analysed. Three proteins, stated to be biomarkers of nephrotic syndrome, could be identified. In order not to contaminate the manuscript pages with resin particles, we have devised a novel harvesting film, by which strong cation and anion exchangers are embedded in ethyl-vinyl acetate foils. It is felt that this technology could be safely applied to other specimens belonging to the Word Heritage.

Introduction

Master i Margarita, the famous novel by Mikhail Bulgakov [1], has been listed among the top 100 books of the twentieth-century. Neither the novel nor its author had an easy life: Bulgakov started its writing in 1928 but burned the manuscript in 1930. He then began working on a second draft that was completed in 1936. Yet the novel appeared in a book form long after his death, in 1967. The reason being that, although Bulgakov was a brilliant mind and a writer of several theater pieces, envious critics (and servants of the Stalin repressive regime) kept giving him bad reviews and forced government censors to prevent publication of any of his work and staging any of his plays. Although in 1936 the major plot lines of this version were in place, Bulgakov kept reshaping and re-elaborating the text anew in four subsequent versions during the years 1936–1940 and he only stopped writing four weeks before his death in 1940.

In a first investigation [2] on ten pages of his original manuscript, by adopting a mixture of strong cation exchanger beads admixed with crushed C₈ resins, loosely layered over the margins, we detected significant amounts of morphine, likely used by the author as a pain reliever. Two unexpected events after this publication spurred the present investigation. One was the observation by an English scientist stating that our technique, which would surely leave part of the resin trapped into the foils fibres, could hardly be adopted for exploring precious archaeological material stored in a museum. Taking this criticism into account, we have devised a novel harvesting methodology, presented here for the first time. The second observation was made by Prof. Serena Vitale, expert on Russian literature and author of a famous novel on the life of Puskin [3]. She claimed that, since there was no historical trace of Bulgakov ingesting morphine later in his life, the drug could have been left by the NKVD (the dreaded secret police of Stalin, today called KBG), who had sequestered the original manuscript and stored it in their archives for some decades. Since it is known that, in the last few years of his life, the author was suffering from a severe form of nephrosis (an inherited kidney disorder) that eventually took him to his grave, we thus wondered if we could find traces of proteins in these pages to potentially identify biomarkers of his renal pathology. Having had access to his final manuscript, we have explored the surface of another ten original pages (different from those already analysed in ref. 2) via the novel methodology here reported, enabling the harvest of surface material without damaging these precious documents, which pertain to the world heritage. The results of this investigation are given below.