Publications by authors named "Gleb Zilberstein"

27 Publications

  • Page 1 of 1

New baits for fishing in cultural heritage's Mare Magnum.

J Proteomics 2021 03 14;235:104113. Epub 2021 Jan 14.

Spectrophon Ltd, Oppenheimer 7, Rehovot 7670107, Israel.

We describe here a modern tool for exploring documents pertaining to the world Cultural Heritage while avoiding their contamination or damage. Known under the acronym EVA, it consists of a plastic foil of Ethylene Vinyl Acetate studded with strong cation and anion resins admixed with C and C hydrophobic beads. When applied to any surface such foils can harvest any type of surface material, which is then eluted and analyzed via standard means, such as GS/MS (typically for metabolites), MS/MS (for peptide and protein analysis), X-ray (for elemental analysis). We briefly review here a number of past data, such as screening of original documents by Bulgakov, Chekov, Casanova, Kepler, while dealing in extenso with very recent data, pertaining to Orwell and Stalin and analysis of the skin of an Egyptian mummy. The technique was also successfully applied to paintings, such as the Donna Nuda at the Hermitage in St. Petersburg, attributed to Leonardo and his school. This novel methodology represents a formidable tool for exploring the past life of famous authors, scientist and literates in that it can detect traces of their pathologies and even drug consumption left by saliva and sweat traces on their original hand-written documents.
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http://dx.doi.org/10.1016/j.jprot.2021.104113DOI Listing
March 2021

Stalin's "black dog": a postmortem diagnosis.

Anal Bioanal Chem 2020 Nov 2;412(28):7701-7708. Epub 2020 Sep 2.

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, 20131, Milan, Italy.

Undoubtedly, the two leaders who were under enormous pressure during World War II (WWII) were Winston Churchill and Joseph Stalin' since their respective countries had to sustain most of the war weight, at least in Europe. Lord Moran recounted in his memoir Winston Churchill: The Struggle for Survival that he had diagnosed a middle-aged Churchill with bipolar disorder. Churchill himself often referred to his periods of intense and prolonged depression as his "black dog." On the contrary, not much is known about Stalin's mental conditions, although in 1927 the neurologist V. M. Bekhterev, the day prior to his sudden death, upon a long examination of the leader's mental status, declared that he had found him affected by paranoia. No chemical evidence via clinical chemistry analyses was provided for the two leaders, though. We have had access to the collection of books (stored in the Russian Government Archive of Social and Political History, RGASPI, of the former Institute of Marxism and Leninism under the Central Committee of the USSR Communist Party) that Stalin was reading during WWII, with pages containing personal annotations on the margins. Upon harvesting surface material via EVA disks (ethylene-vinyl acetate studded with strong cation and anion exchangers and C-C resins) and instrumental analysis via X-ray photoelectron spectroscopy, we detected lithium levels (~ 100 ± 8 ng/cm) compatible with those present in the sweat and/or saliva of patients treated with lithium salts for curing bipolarity and paranoia or probably gout. These data are the first clear indication that Stalin was under cure for this pathology.Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02914-zDOI Listing
November 2020

Surface analysis of ancient parchments via the EVA film: The Aleppo Codex.

Anal Biochem 2020 09 7;604:113824. Epub 2020 Jul 7.

Department of Chemistry, Materials and Chemical Engineering ''Giulio Natta'', Politecnico di Milano, Via Mancinelli 7, Milano, 20131, Italy. Electronic address:

The margins of several pages of the Aleppo codex have been found to be corroded and contaminated by diffuse maculae. In order to understand the origin of this decay these margins have been analysed by applying EVA (ethylene vinyl acetate plastic embedded with strong cation and anion exchangers and mixed with C and C hydrophobic resins) diskettes for harvesting surface material. The captured compounds have been eluted, digested with trypsin and analysed by nano-HPLC-MS. Three major strains of Aspergillus have been identified, namely Aspergillus fumigatus, Aspergillus pseudoglaucus, Aspergillus amstelodami, together with a lactobacillus strain and human keratins. The novelty of this investigation is that for the first time the EVA technology has been applied to ancient parchments in the absence of mechanical deformation or distortion that could be induced if there had been water exchange between the EVA diskettes and the parchment. These findings should help curators to find suitable restoration protocols for these precious documents belonging to the world Cultural Heritage.
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http://dx.doi.org/10.1016/j.ab.2020.113824DOI Listing
September 2020

"1984": What Orwell could not predict. Proteomic analysis of his scripts.

Electrophoresis 2020 11 9;41(21-22):1931-1940. Epub 2020 Jun 9.

Department of Chemistry, Materials and Chemical Engineering, Milano, Italy.

George Orwell, fighter for the Republican Army during the Spanish Civil War, was shot through the throat by a sniper on 20th May 1937 and nearly killed. After receiving only a summary external treatment, on the 29th, he was cured in a Barcelona hospital where he was infected by the Koch bacillus. After fleeing from Spain on 23rd June 1937, he repaired to his cottage in Wallington, Hertfordshire, wherefrom he wrote a letter to Sergey Dynamov, Editor of Soviet journal "Foreign Literature." This typewritten letter was analyzed by application of five EVA strips (ethylene vinyl acetate studded with strong cation and anion and with C and C resins; four on the corners and one over his signature), searching for biological traces. Upon elution of the captured biologicals, trypsin digestion and Orbitrap Fusion trihybrid mass spectrometer analyses, three of the five strips yielded clear traces of six unique proteins (via proteotypic peptides) of the tuberculosis bacterium. Additionally, MALDI TOF analysis of saliva of a tuberculosis patient and the EVA strip eluates gave a spectrum of 14 peptide bands (Mr 2700 to 6700 Da range) coincident between the two samples, thus, fully confirming Orwell's pathology. These results are attributed to saliva traces on Orwell's fingertips and to the fact that the letter was written on 2nd July 1937, when Orwell's pathology was at its peak.
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http://dx.doi.org/10.1002/elps.202000063DOI Listing
November 2020

EVA Technology and Proteomics: A Two-Pronged Attack on Cultural Heritage.

J Proteome Res 2020 08 17;19(8):2914-2925. Epub 2020 Jun 17.

Spectrophon Ltd., Oppenheimer 7, Rehovot 7670107, Israel.

A novel way for exploring the world's cultural heritage in the absence of damage or contamination (such as removing pigments in paintings or chipping away pieces of bones) of the items under investigation is here reported, called the EVA technique. It is based on films of ethylene vinyl acetate (EVA) impregnated with strong anion and cation exchangers, admixed with hydrophobic resins, C and C. When in contact with any surface these films can harvest nanomoles of macromolecules (proteins and DNA) as well as metabolites, which can then be identified by standard instrumentation. Some important applications are reported, such as the findings of the renal pathology and assumption of morphine in the original manuscript of Master I Margarita by Bulgakov, the presence of TBC bacterium in Chekhov's shirt and in a letter by Orwell, the and anthrax bacteria in the death registries of Milan's lazaretto in the 1630 plague bout, as well as ample traces of five metals in Kepler's manuscripts, suggesting his potential practice of alchemy. Also, in the pages of the Memoirs of Casanova, although the gonorrhea bacterium could not be found, spots of HgS could be measured, suggesting its use for curing the disease. A family of EVA films is described, enlarging its use to dedicated applications, such as the capture of drugs of abuse in the pages of famous writers and even in the paintings of fauvists. It is hoped that the present methodology could open the doors of museums, state archives, and private collections for detecting biological traces left by artists, literates, and men of culture in their masterpieces.
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http://dx.doi.org/10.1021/acs.jproteome.0c00080DOI Listing
August 2020

De re metallica. Johannes Kepler and alchemy.

Talanta 2019 Nov 26;204:82-88. Epub 2019 May 26.

Department of Chemistry, Materials and Chemical Engineering ''Giulio Natta'', Politecnico di Milano, Via Mancinelli 7, Milano, 20131, Italy. Electronic address:

The application of analytical chemistry to the exploration of the World Cultural Heritage represents a major challenge in that most protocols and strategies are invasive and require micro-sampling. We report a novel methodology for harvesting material deposited on the surface of ancient documents while avoiding their damage or contamination. The technology here described relates to the capture of metals on these specimens. It is based on the use of plastic films (ethylene vinyl acetate, EVA) impregnated with different metal chelators (sodium 2,3-dimercapto-1-propanesulfonate, DMPS, meso-2,3-dimercaptosuccinic acid, DMSA and ethylene diamino tetra acetic acid, EDTA, as calcium salt), for harvesting from surfaces of different supports potential traces of metals therein deposited. The EVA film technology has been used to explore the pages of a manuscript written by Kepler concerning the movements of the moon and catalogued under the title "Hipparchus" at the Archives of the Russian Academy of Sciences (St. Petersburg branch). The EVA-based chelating diskettes were able to capture very significant amounts of different metals, namely: Au, Ag, Hg, As, Pb, suggesting that Kepler, well known as astronomer, astrologist, mathematician and Lutheran theologian, might have started practicing alchemy, a pseudo-chemical science he had learned from his colleague Tycho Brahe in Prague.
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http://dx.doi.org/10.1016/j.talanta.2019.05.094DOI Listing
November 2019

Leonardo's Donna Nuda unveiled.

J Proteomics 2019 09 16;207:103450. Epub 2019 Jul 16.

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Electronic address:

The painting "Donna Nuda" by Leonardo was acquired by Catherine II (the Great) from the R. Walpole collection, Houghton Hall, England, in 1779 for the Hermitage in St. Petersburg. By exploiting the EVA film technology (ethylene vinyl acetate polymer embedded with strong cation and anion exchangers and with C and/or C resins) we have explored the surface of the painting in order to ascertain the techniques used in its drawing. Five EVA films were affixed on the body and on the landscape for 60 min. Upon elution from the recovered films, the harvested material was analyzed by gas-chromatography/mass spectrometry as well as by liquid chromatography/mass spectrometry. "Tempera grassa" (consisting of linseed oil admixed with egg yolk) was used in the entire painting. The surface was then protected by a layer of conifer resin. It is hypothesized that access to the layer underneath the protective layer was obtained via micro-cracks on the conifer resin itself. Rosemary oil was used as diluent to slow down the drying process and so to perform the glazing technique, thus obtaining the "aerial perspective" in correspondence of the landscape. To our reckoning, this is the first time in which a Leonardo painting is analyzed in depth but also in which his artistic technique is deciphered via modern techniques for exploring Cultural Heritage. The EVA film technology might be used for ascertaining the authenticity of paintings and uncover frauds. SIGNIFICANCE STATEMENT: Leonardo da Vinci was the most famous Italian polymath of the Renaissance and one of the most important innovators of his time. He was the author of several important artworks such as "La Gioconda", but he also painted the "Donna Nuda" conserved at the Hermitage Museum. Although some attempts permitted the identification of part of the materials used by Leonardo, to date no analytical investigations were able to fully characterize and decipher the recipes. We explored the surface of the "Donna Nuda" painting through a non-invasive approach that uses a functionalized film to adsorb nano-scopic amounts of materials that were then analyzed by mass spectrometry. This method has the potential to revolutionize the approaches used to analyze cultural heritage.
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http://dx.doi.org/10.1016/j.jprot.2019.103450DOI Listing
September 2019

What Sherlock sorely missed: the EVA technology for cultural heritage exploration.

Expert Rev Proteomics 2019 06 30;16(6):533-542. Epub 2019 May 30.

c Department of Pharmaceutical Sciences , Università degli Studi di Milano , Milano , Italy.

: Capture of proteins and metabolites from Cultural Heritage (paintings, manuscripts, parchment etc.) has been done in the past via surface scraping and erasing, a method discouraged. The EVA (ethylene vinyl acetate) method consists of a plastic polymer in which strong cation and anion resins, admixed with C and/or C, are embedded. : We review here the findings on different items stored in public libraries and archives: (a) the original manuscript of the novel Master y Margarita by Bulgakov; (b) the death registries of the lazaretto in the 1630 Milano plague; (c) the shirt worn by A. Chekhov in his death bed; (d) Kepler's script on Hipparchus (in St. Petersburg National Archives); (e) the Memoirs of G. Casanova. : The technique here surveyed appears to be a unique tool enabling exploration of any document stored in public archives, museum and private collections without damaging or contaminating the items under analysis. The amounts harvested from any surface are very minute, yet sufficient for analysis via advanced mass spectrometry instrumentation, thus permitting the identification of all captured material. It is hoped that the present review will stimulate the scientific community to adopt it for projects pertaining to Cultural Heritage.
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http://dx.doi.org/10.1080/14789450.2019.1624164DOI Listing
June 2019

Il n'y a pas d'amour heureux pour Casanova: Chemical- and bio-analysis of his Memoirs.

Electrophoresis 2019 12 15;40(23-24):3050-3056. Epub 2019 Apr 15.

Department of Chemistry, Materials and Chemical Engineering ''Giulio Natta'', Politecnico di Milano, Milano, Italy.

The original manuscript of Casanova's Memoirs is stored at the Bibliothèque Nationale de France in Paris. We have gained access to it and explored the surfaces of chapters one and two (via the ethylene vinyl acetate [EVA] film technology, i.e., of diskettes of ethylene vinyl acetate with embedded strong cation and anion exchangers and C8 resins) in search of potential diseases of the author, especially of the gonorrhea bacterium, since Casanova reported that he had several bouts of this pathology along his adventurous life. Although the bacterium was not found, we have detected high levels of HgS as red spots along the lines of the manuscript, suggesting that Casanova was using this chemical as a cure for his venereal disease. Additionally, among the several bacteria identified on the surface via mass spectrometry, we could detect traces of Streptococcus uberis, a typical animal infection, found also in humans, together with a few strains of Lactobacilli, probably present in his saliva. The EVA film technology appears to open new horizons for investigating the world Cultural Heritage.
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http://dx.doi.org/10.1002/elps.201800505DOI Listing
December 2019

Noninvasive wearable sensor for indirect glucometry.

Electrophoresis 2018 09 30;39(18):2344-2350. Epub 2018 Apr 30.

Department of Chemistry, Politecnico di Milano, Milano, Italy.

A noninvasive mini-sensor for blood glucose concentration assessment has been developed. The monitoring is performed by gently pressing a wrist or fingertip onto the chemochromic mixture coating a thin glass or polymer film positioned on the back panel of a smart watch with PPG/HRM (photoplethysmographic/heart rate monitoring sensor). The various chemochromic components measure the absolute values of the following metabolites present in the sweat: acetone, acetone beta-hydroxybutirate, aceto acetate, water, carbon dioxide, lactate anion, pyruvic acid, Na and K salts. Taken together, all these parameters give information about blood glucose concentration, calculated via multivariate analysis based on neural network algorithms built into the sensor. The Clarke Error Grid shows an excellent correlation between data measured by the standard invasive glucose analyser and the present noninvasive sensor, with all points aligned along a 45-degree diagonal and contained almost exclusively in sector A. Graphs measuring glucose levels five times a day (prior, during and after breakfast and prior, during and after lunch), for different individuals (males and females) show a good correlation between the two curves of conventional, invasive meters vs. the noninvasive sensor, with an error of ±15%. This novel, noninvasive sensor for indirect glucometry is fully miniaturized, easy to use and operate and could represent a valid alternative in clinical settings and for individual, personal users, to current, invasive tools.
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http://dx.doi.org/10.1002/elps.201700424DOI Listing
September 2018

Anton Chekhov and Robert Koch Cheek to Cheek: A Proteomic Study.

Proteomics 2018 05;18(9):e1700447

Department of Chemistry, Materials and Chemical Engineering ''Giulio Natta'', Politecnico di Milano, Milano, Italy.

Five different letters and post cards as well as the shirt worn by Anton Chekhov on his death bed, stored in the State Literary-Memorial Museum-Reserve A. P. Chekhov Melikhovo (nearby Moscow), have been analyzed by applying EVA (an ethyl vinyl acetate foil studded with crushed strong anion and cation exchangers and with C resins) diskettes to these surfaces. Three different eluates (under acidic and basic conditions and with acetonitrile) were analyzed by high resolution mass spectrometry. The environmental microbiota present on samples and the Mycobacterium tuberculosis strain were described by a meta-proteomics approach. Eight identified M. tuberculosis proteins confirmed the presence of the bacterium and the cause of Chekhov's death, in addition to several sequenced peptides belonging to other bacterial species. The human plasma proteins and human keratins, detected on a tiny blood spot on the shirt, demonstrated the power of the combined approach.
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http://dx.doi.org/10.1002/pmic.201700447DOI Listing
May 2018

Of mice and men: Traces of life in the death registries of the 1630 plague in Milano.

J Proteomics 2018 05 3;180:128-137. Epub 2018 Jan 3.

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Electronic address:

The death registries of the plague epidemic of 1630, stored at the Archivio di Stato of Milano, have been interrogated via the EVA film technology (ethyl vinyl acetate film studded with crushed strong anion and cation exchangers as well as C resins). The EVA diskettes have been left in contact with the lower right margins of 11 different pages pertaining to the peak months of the raging disease (June through end of September) for 60-90min and then the captured material, after elution and digestion, analysed by mass spectrometry. The main findings: 17 Yersiniaceae family proteins, 31 different human keratins, 22 unique mouse keratins, about 400 peptides from different bacterial strains, 58 human tissue proteins and 130 additional mouse and rat tissue proteins. In addition, >60 plant proteins (notably potato, corn, rice, carrot and chickpeas), likely representing the meagre meals of the scribes, contaminating the pages, were detected. The significance of these unique findings is amply illustrated in the body of the article.

Significance: Archivists, historians, librarians usually explore the texts of ancient and modern manuscript in order to extract the meaning of the writing and understand the mood, feelings, political, philosophical and/or religious ideas therein expressed by the authors. With the present EVA methodology (the only one, at present, able to access our Cultural Heritage without damaging or contaminating it) we interrogate, instead, the support, be it paper, parchment, wood panel, cloth, canvas and the like, in order to extract invisible data, such as the presence of drugs, medicaments, infectious pathogens, human and environmental contaminants. Metabolites, proteins and peptides thus captured are then analysed via mass spectrometry. The unique data mined by this technology should considerably enlarge the (so far) restricted horizon of the writing exploration and add new insight on the environmental conditions in which such documents were produced as well as, importantly, on the health/pathological conditions of the authors. It is believed that the present technology, as here reported, will become the officially accepted one for exploring the world Cultural Heritage.
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http://dx.doi.org/10.1016/j.jprot.2017.11.028DOI Listing
May 2018

A miniaturized sensor for detection of formaldehyde fumes.

Electrophoresis 2017 09 20;38(17):2168-2174. Epub 2017 Jun 20.

Department of Chemistry, Materials and Chemical Engineering ''Giulio Natta'', Politecnico di Milano, Milano, Italy.

A miniaturized chemical sensor is here described for the analysis of environmental pollutants (VOC: volatile organic chemicals). It is used for remote detection of formaldehyde (FA) fumes in the atmosphere, and is based on the redox reaction between FA and silver nitrate. The sensor is worn as a bracelet and the data acquired are transferred via a Bluetooth channel to a smartphone. A dedicated software transforms the signal from a grey to a color scale. The signal response has been assessed over low (20 to 120 ppb) as well as higher (1-15 ppm range) levels. The sensor has been applied to monitor potential FA fumes of some artwork in the Summer Palace in Beijing and the modifications induced by FA treatment on a precious Stradivarius violin. The performance of this novel sensor is compared with a commercial apparatus widely adopted, namely the Honeywell MultiRAE Lite wireless portable multi-gas monitor (pumped model).
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http://dx.doi.org/10.1002/elps.201600559DOI Listing
September 2017

Method for Noninvasive Analysis of Proteins and Small Molecules from Ancient Objects.

Anal Chem 2017 03 3;89(6):3310-3317. Epub 2017 Mar 3.

Department of Chemistry, Materials and Chemical Engineering "'Giulio Natta"', Politecnico di Milano , Via Mancinelli 7, Milano 20131, Italy.

Proteins and small molecules from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all present-day protocols and strategies for the analysis of ancient samples are often invasive and require microsampling. Here, we present a new method for the noninvasive analysis of proteins and small molecules: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C resins, for interacting with both proteins and small molecules present on the surface of the objects, followed by LC-MS/MS analysis. The new method was fully validated for the determination of both proteins and small molecules on several types of supports, showing excellent analytical performances such as, for example, R of the calibration curve of 0.98 and 0.99 for proteins and small molecules, low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the analytical procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally, the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require microsampling and the proteins/small molecules extraction can be performed directly in situ, leaving the object unchanged and intact.
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http://dx.doi.org/10.1021/acs.analchem.6b03722DOI Listing
March 2017

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

J Proteomics 2017 01 29;152:102-108. Epub 2016 Oct 29.

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jprot.2016.10.019DOI Listing
January 2017

Maestro, Marguerite, morphine: The last years in the life of Mikhail Bulgakov.

J Proteomics 2016 Jan 3;131:199-204. Epub 2015 Nov 3.

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. Electronic address:

Unlabelled: The manuscript pages of the final draft of Master i Margarita, the masterpiece by Mikhail Bulgakov, written in the last four years of his life (1936-1940), have been treated with a mixture of chromatographic beads, namely a strong cation exchanger and a C8 resin. Potential substances captured by the beads, after harvesting them, were eluted with a mixture of isopropyl alcohol, dichloromethane and ammonium hydroxide and the eluate subjected to GC-MS analysis in order to detect the presence, if any, of drugs, due to the fact that the writer suffered intense pains caused by an inherited nephrotic syndrome. Indeed all the pages under investigation (a total of ten, taken at random among 127 foils) contained traces of morphine, from as little as 5 up to 100ng/cm(2). In addition to the intact drug, we could detect one of its metabolites, namely 6-O-acetyl morphine. The significance of these findings in terms of a possible improvement of the novel and in terms of drug use (or abuse) in the modern world is discussed and evaluated.

Biological Significance: The extraction of metabolites/proteins from the surface of the original manuscript pages of Bulgakov masterpiece Master i Margarita has permitted to monitor his health state and intake of medicaments over the last four years of his life. We have ascertained that: (1) he was assuming large doses of morphine as pain killers; (2) he was affected by a nephrotic syndrome, since we could identify three proteins known as biomarkers of this pathology. The double extraction procedure here reported could open up a novel field of investigation of (relatively) ancient manuscripts for metabolome/proteome analysis on the health status of the writer/artist.
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http://dx.doi.org/10.1016/j.jprot.2015.11.002DOI Listing
January 2016

Third generation of focusing: gel matrices with immobilized cation gradients.

Electrophoresis 2010 Jun;31(11):1747-53

Cleardirection Ltd., Rehovot, Israel.

A novel method is reviewed here for separation of polyanions, based not on conventional zone electrophoretic means, but on a "steady-state" process by which said polyanions are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such polyanions matches the surrounding charge density of the matrix, they stop migrating and remain stationary, as typical of steady-state separation techniques. This technique has been successfully applied to SDS-protein micelles, DNAs, RNAs and heparins, with remarkable separations, often much superior than those obtained in conventional techniques. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced charge densities. This technique resembles a classical IEF process, with the proviso that the polyanions cannot be applied at any position along the gel matrix, but only at the point of low (or zero) charge density. The merits and limits of the technique are assessed.
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http://dx.doi.org/10.1002/elps.200900602DOI Listing
June 2010

Focusing of low-molecular-mass heparins in polycationic polyacrylamide matrices.

Anal Chem 2009 Aug;81(16):6966-71

Cleardirection Ltd., 4 Pekeris St., Rehovot 76702, Israel.

A novel method for separation of low-molecular-mass heparins is reported here, on the basis of migrating the polyanionic heparins in a polycationic polyacrylamide gel, made by incorporating a gradient of positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated either in linear or nonlinear gradients of positive charges, thus modulating at whim the separation power. This allows the polydisperse heparins to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization. It is shown that the separations obtained are a complex function of both size and charge distribution along the oligosaccharide chains. This novel methodology represents a marked improvement over existing techniques and appears to hold promise for applications in screening of commercial lots of heparins, also in view of possible presence of contaminants, such as those recently detected in imported heparins.
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http://dx.doi.org/10.1021/ac901050qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846550PMC
August 2009

Steady-state electrophoresis of RNA against a gradient of cationic charges in a polyacrylamide matrix.

Electrophoresis 2009 Nov;30(21):3696-700

Cleardirection Ltd., Rehovot, Israel.

A novel method for separation of RNA fragments is reported here, based on migrating the polyanionic RNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations are typically performed in a 0-10 mM, pK 10.3 Immobiline gradient under denaturing conditions (6 M urea). In the 100-1000 bp length, it is shown that separations of RNA are optimal and very sharp bands can be obtained, in comparison with conventional electrophoresis, due to the "focusing" effect originated by the charge balancing between the positively charged gel matrix and the negatively charged RNA species. Excellent separations are also obtained from micro-RNAs, single-stranded RNA molecules of 21-23 nucleotides in length, which appear to regulate gene expression in animal and plant tissues. As a third example, 2-D runs in control and polycationic gels are shown. Under native conditions, RNAs are not aligned in a diagonal, suggesting that molecular shape has a strong influence on the interaction between RNA and the charged gel matrix. Thus, 2-D runs in cationic matrices might be exploited for structural studies of RNA molecules.
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http://dx.doi.org/10.1002/elps.200900321DOI Listing
November 2009

High-resolution separation of peptides by sodium dodecyl sulfate-polyacrylamide gel "focusing".

Electrophoresis 2008 Apr;29(8):1749-52

Cleardirection Ltd., Rehovot, Israel.

As a follow-up of our previous report (Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a refinement of the method for separation of peptides in the small to medium M(r) range (0.5-10 kDa) is here reported, based on a shallow gradient of immobilized positive charges (0-10 mM) onto a minimally sieving polyacrylamide gel matrix (4%T, 2.5%C). Unlike conventional SDS-PAGE, which rarely can achieve the separations of polypeptide chains below a critical value of 10 kDa, the present method can be fine-tuned to perform such separations even down to a size of only 500 Da. In the case of larger fragments, the major peptide zones are shown, under microscope observation, to be composed by envelopes of bands as narrow as 20-100 microm, spaced at regular intervals of 100-150 microm. It is hypothesized that such larger peptides could form complexes with rather small SDS micelles and that such peptide-SDS complexes could differ in charge by just a single negative charge.
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http://dx.doi.org/10.1002/elps.200700625DOI Listing
April 2008

DNA separation methodology based on charge neutralization in a polycationic gel matrix.

Anal Chem 2008 Jul 21;80(13):5031-5. Epub 2008 Mar 21.

Cleardirection Ltd., 4 Pekeris Street, Rehovot 76702, Israel.

A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e., in a gel containing a constant level of positive charges from anode to cathode). In this last case, separation is still obtained due to differential charge modulation of the various DNA fragments. In the 100-1000-bp length, it is shown that separation can be obtained even for fragments differing in length by <0.5%, as shown in the splitting of a 656- and 659-bp doublet, that could not be resolved by conventional polyacrylamide gels. In the 10-100-bp range, it is shown that the present method can resolve single nucleotide polymorphisms, i.e. fragments of identical number of nucleotides but differing by one base substitution. In this last case, separations are obtained only in gradient gels containing a much steeper gradient of charges (0-20 mM Immobiline pK 10.3 and pK 12, as opposed to gradients of only 2-4 mM positive charges for larger size fragments). This novel methodology represents a marked improvement over existing techniques and appears to hold promises for applications in diverse fields, such as molecular biology, forensic medicine, and genetic screening.
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http://dx.doi.org/10.1021/ac800095eDOI Listing
July 2008

SDS-PAGE focusing: preparative aspects.

Anal Chem 2007 Nov 10;79(22):8624-30. Epub 2007 Oct 10.

Cleardirection Ltd., 4 Pekeris Street, Rehovot 76702, Israel.

As a followup of our previous report (Zilberstein, G.; Korol, L.; Antonioli, P.; Righetti, P. G.; Bukshpan, S. Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a novel method is here reported for small-scale prefractionation of complex protein mixtures, for subsequent proteome analysis, based on mass separation of SDS-protein micelles not in a gel matrix, but in liquid cationic polymers assembled in a multicompartment electrolyzer (MCE) in a stepwise fashion at discrete and increasing levels of positive charges (from 3 to 28 mM), the neighboring chambers being separated by neutral agarose membranes. Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method of SDS-PAGE focusing exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path and trapped into different compartments of the MCE device via interaction (and subsequent charge neutralization) with cationic polymers of fixed (but increasing from chamber to chamber from cathode to anode) charge density. Minimization of migration of the liquid cationic polymers is obtained via use of low voltage and by arranging for a buffer conductivity gradient along the migration path. The present setup has the advantage of high protein recoveries (up to 90%) without any contamination from ungrafted monomers and catalysts, as occurring in proteins recovered by passive elution from gel matrixes. Additionally, resolution can be fine-tuned by selecting cationic polymers of varying charge density in microstep increments. The cationic polymers, of desired charge density and proper viscosity, are prepared by standard polymerization conditions, can be easily precipitated and washed free of monomeric contaminants, and stored in a dry form for subsequent use.
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http://dx.doi.org/10.1021/ac701598yDOI Listing
November 2007

SDS-PAGE under focusing conditions: an electrokinetic transport phenomenon based on charge compensation.

Anal Chem 2007 Feb;79(3):821-7

Cleardirection Ltd., 4 Pekeris Street, Rehovot 76702, Israel.

A novel method is reported for mass separation of proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method, called "SDS-PAGE focusing", exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such complexes matches the surrounding charge density of the matrix, the SDS-protein complex stops migrating and remains stationary, as typical of steady-state separation techniques. As a result of this mechanism, the proteins are separated in an unorthodox way, with the smaller proteins/peptides staying closer to the application point and larger proteins migrating further down toward the anodic gel end. This results in a positive slope of the Mr vs migration plot, vs a negative slope in conventional SDS-PAGE. Moreover, such a plot is linear (by design), whereas in standard SDS-PAGE it is semi- or even double logarithmic. Particularly advantageous appears the ability of the present method to fine-tune the separation of small-size fragments and tryptic digests, where conventional SDS-PAGE usually fails. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced Mr values, down to a limit of approximately 150 Da. This increases the resolution by at least 1 order of magnitude as compared with standard SDS-PAGE, where for a proper separation of two adjacent species, an Mr increment of approximately 3000 Da is needed.
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http://dx.doi.org/10.1021/ac0615091DOI Listing
February 2007

Parallel isoelectric focusing II.

Electrophoresis 2004 Nov;25(21-22):3643-51

Protein Forest Inc., Rehovot, Israel.

A miniature electrophoretic device is developed on the basis of a new isoelectric focusing (IEF) method, namely parallel isoelectric focusing. We report here the theory and the results of operation of a new parallel isoelectric device (PID). The main advantages and limitations of the method are discussed for miniaturization purposes. It is shown that the method guarantees the fast and complete separation of any complex protein mixtures under acceptable conditions, such as voltage source, temperature, size of the device, and separation process duration. It is shown that the main problem of PID miniaturization is the buffer design, and the relation between Immobiline buffer capacity and solution buffer capacity. The main experimental limitation of PID resolution is protein sensitivity to pH changes.
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http://dx.doi.org/10.1002/elps.200406117DOI Listing
November 2004

Parallel isoelectric focusing chip.

Proteomics 2004 Sep;4(9):2533-40

Protein Forest, Rehovot, Israel.

Fast isoelectric focusing (IEF) is becoming a key method in modern protein analysis. We report here the theory and experimental results of new parallel isoelectric devices (PID) for fast IEF. The main separation tool of any PID is a dielectric membrane with conducting channels filled by immobiline gels of varying pH. The pH value of the surrounding aqueous solution is not equal to the pH of any of the channels. The membrane is held perpendicular to the applied electric field. Proteins are collected (trapped) in the channels whose pH values are equal to the pI of the proteins. The fast particle transport between different channels takes place due to convection in the aqueous solution. We developed a mathematical model for PID. Experiment duration is shown to be proportional to the number of different bands N (the peak capacity in standard IEF) in contrast with N(2) for usual IEF devices. This model was validated with experimental results. Parallel IEF accelerates the fractionation of proteins by their pI values (down to several minutes) allowing a more desirable collection efficiency to be achieved. The main theoretical limitation of PID resolution is the sensitivity of proteins to pH change due to the Coulomb blockade effect. The existence of a minimal pH change deltapH(min) for each type of protein is shown: deltapH(min) approximately r(-1) for globular molecules with radius r.
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http://dx.doi.org/10.1002/pmic.200300794DOI Listing
September 2004

Parallel processing in the isoelectric focusing chip.

Electrophoresis 2003 Nov;24(21):3735-44

Protein Forest Inc., Rehovot, Israel.

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.
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http://dx.doi.org/10.1002/elps.200305663DOI Listing
November 2003

Nonlinear electrophoresis of point-like particles--is it possible?

Electrophoresis 2002 Aug;23(16):2626-34

Protein Forest (Israel) Inc., 4 Pekeris Street, Weizmann Science Park, 76702 Rehovot, Israel.

A new universal method for the generation of nonlinear electrophoretic mobility of a packet of any particles is suggested. The method is based on the investigation of particle packet dynamics under the influence of an external force. The system under consideration is a homogeneous and isotropic medium with traps for these particles. Packet dynamics is described by a linear diffusion equation. The measured packet parameters are the position and the velocity of a packet maximum. It is shown that these parameters are nonlinear in the external field under definite limitations on the trap properties. This statement is proved both theoretically and experimentally for the simple model of diffusive substrate, the so-called comb structure. The prospects of designing new supporting substrates (microfluidic systems) with a nonlinear response are discussed.
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http://dx.doi.org/10.1002/1522-2683(200208)23:16<2626::AID-ELPS2626>3.0.CO;2-5DOI Listing
August 2002