Advanced techniques in clinical practice: Use of lab-on-a-chip electrophoresis and other methods in protein profiling

JMB 2009; 28 (4) DOI: 10.2478/v10011-009-0026-7 UDK 577.1 : 61 ISSN 1452-8258 JMB 28: 274 –278, 2009 Original paper Originalni nau~ni rad ADVANCED TECHNIQUES IN CLINICAL PRACTICE: USE OF LAB-ON-A-CHIP ELECTROPHORESIS AND OTHER METHODS IN PROTEIN PROFILING NAPREDNE TEHNIKE U KLINI^KOJ PRAKSI: ELEKTROFOREZA TIPA LABORATORIJA NA ^IPU I DRUGE METODE ZA PROFILISANJE PROTEINA Olgica Tren~evska1, Vasko Aleksovski2, Kiro Stojanoski1 1Institute of Chemistry, Faculty of Natural Sciences, Sts. Cyril and Methodius University, Skopje, Republic of Macedonia 2Neurology Clinic, Faculty of Medicine, Sts. Cyril and Methodius University, Skopje, Republic of Macedonia Summary: Proteins in clinical practice are analyzed as important parameters in the determination and treatment of different diseases. The scopes of the analyses are mainly concentrated in two levels – analyses of the complete protein profile, or determination of an isolated protein. In this work, despite of the use of conventional methods, mainly electrophoresis, new techniques have been implemented in protein analyses. Lab-on-a-chip is an electrophoretic technique that, when optimized, provides analyses of the total protein profile. When normal samples are compared to samples obtained from patients with different neurological diseases, characteristic patterns can be noted. Also, correlation and comparison can be made between the newly developed microchip electrophoresis method and the results obtained using the conventional techniques. When an analysis of a specific protein is necessary, mass spectrometry has proven to give best results, in both the selectivity and specificity of analyses. It is believed that cystatin C is a potential biomarker in neurological diseases; therefore, the mass spectrometry method has been developed in order to obtain qualitative and quantitative analyses of biological fluids. Using the developed method of mass spectrometry immunoassay (MSIA), cystatin C was easily isolated and analyzed, obtaining complete analysis within minutes. The resulting mass spectra revealed various levels of cystatin C isoforms in serum and CSF samples. Keywords: cystatin C, lab-on-a-chip electrophoresis, MALDI-TOF-MS, MSIA, neurological diseases, protein profiling Address for correspondence: Olgica Tren~evska Institute of Chemistry Faculty of Natural Sciences, Sts. Cyril and Methodius University Arhimedova 5, PO Box 162 1001 Skopje, Republic of Macedonia phone no: + 389 70 54 24 60 fax no: + 389 2 3226 865 e-mail: olja.trencevskaªgmail.com Kratak sadr`aj: U klini~koj praksi, proteini se analiziraju kao parametri va`ni za odre|ivanje i le~enje razli~itih bolesti. Postoje dva opsega analiza: analize kompletnog proteinskog profila ili odre|ivanje izolovanog proteina. U ovom radu, pored kori{}enja uobi~ajenih metoda, pre svega elektroforeze, u analizu proteina uklju~ene su nove tehnike. Laboratorija na ~ipu je elektroforetska tehnika koja, optimizovana, omogu}ava analize ukupnog proteinskog profila. Kada se normalni uzorci uporede sa uzorcima dobijenim od pacijenata s razli~itim neurolo{kim oboljenjima, postaju uo~ljivi karakteristi~ni obrasci. Tako|e, mogu}e je uspostaviti korelaciju i pore|enje izme|u nedavno razvijenih metoda »mikro~ip« elektroforeze i rezultata dobijenih kori{}enjem uobi~ajenih tehnika. Kada je neophodna analiza specifi~nog proteina, masena spektrometrija je dala najbolje rezultate, {to se ti~e selektivnosti kao i specifi~nosti analiza. Postoji uverenje da je cistatin C potencijalni biomarker za neurolo{ka oboljenja, stoga je razvijen metod masene spektrometrije kako bi se postigle kvalitativne i kvantitativne analize biolo{kih te~nosti. Pomo}u metode imunoeseja masenom spektrometrijom (MSIA), cistatin C je lako izolovan i analiziran a kompletna analiza trajala je nekoliko minuta. Maseni spektri koji su nastali kao rezultat otkrili su razli~ite nivoe izoformi cistatina C u serumu i uzorcima cerebrospinalne te~nosti. Klju~ne re~i: cistatin C, elektroforeza tipa »laboratorija na ~ipu«, MALDI-TOF-MS, MSIA, neurolo{ka oboljenja, profilisanje proteina Introduction Everyday laboratory analyses are done in order to contribute to precise medical diagnosis in patients with different diseases. The techniques developed include methods which are easy to handle, provide results with satisfying selectivity and sensitivity, and are fast enough to give results in a short time. JMB 2009; 28 (4) Proteins can be analyzed in different biological specimens and in two frames – either in general, obtaining an image of the total protein profile or by analysis of a selected protein, which is considered to have a role as a potential biomarker for a certain disease. Both methods contribute to the precise clinical diagnosis and provide physicians with the required information about the patients’ health. Obtaining a complete protein profile is important because information can be provided about the different classes of proteins present in the sample, therefore giving the opportunity to calculate specific indexes that are usually implemented as diagnostic criteria in some diseases (1). On the other hand, there are specific proteins, potential biomarkers, which, when present in the biological fluid, can indicate specific dysfunction (2). In this work, we have used lab-on-a-chip electrophoresis and mass spectrometry in order to analyze real samples (serum and cerebrospinal fluid) from patients with neurological diseases. We have analyzed proteins in two levels – by obtaining total protein profile through optimized lab-on-a-chip electrophoresis (3), and by analyzing a specific protein, cystatin C, an acute phase protein, which is considered to be a potential biomarker in neurological diseases, by a mass spectrometry immunoassay. Miniaturized lab-on-a-chip electrophoresis is a novel technique in protein profile analyses (4). The microchip system includes a system of microchannels through which gel is rushed by applying pressure. The gel contains fluorescent dye which serves as a label; therefore protein detection is done by fluorescence analysis. By running the chip, proteins can be separated according to their size, and, as a result, the protein profile can be obtained. Mass spectrometry is still not widely used in routine clinical practice. However, its importance in analyses of specific proteins as potential biomarkers will lead to its implementation in this field. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a technique that is used to provide a rapid and sensitive profiling method for molecular mass calculation for proteins, as well as for biomarker discovery, from a variety of biological samples. It is considered that cystatin C, the protein found in biological fluids, is a potential biomarker for several neurological diseases, although its role is still not determined (5). By developing novel MSIA methods, using MALDI-TOF-MS in order to analyze cystatin C levels in (...truncated)