One of the biggest challenges for scientists in analyzing proteomic samples is related to the presence of thousand of compounds occurring in a wide concentration range. The complexity of such samples becomes even greater after enzymatic digestion to peptides, as a result it is crucial to develop technologies with extremely good resolving power and with extraordinary sensitivity to tackle such a task. Two dimensional capillary and nano liquid chromatography has reduced the requirement for loading large amounts of samples, which is important for the analysis of low-abundance compounds or available in limited quantities. In our approach we developed a comprehensive two-dimensional liquid chromatography (LC×LC) platform comprised of two capillary C18 columns in both dimensions hyphenated by means of two electronically controlled six-port two-positions nano switching valves. Such a system was operated under stop-flow conditions while orthogonality was ensured by the employment of the different pH in both dimensions. A newly designed software was used for data elaboration and visualization of selected peaks in the 2D plane. Good repeatability of retention times and peak areas was attained, holding promise for an application in real-case studies; current research is devoted to improve and speed up analysis by capillary monolithic columns and to optimize MS detection for search into database and protein identification.
ON-LINE CAPILLARY SCALE COMPREHENSIVE TWO DIMENSIONAL LIQUID CHROMATOGRAPHY SYSTEM FOR THE ANALYSIS OF PROTEOME SAMPLES
SOMMELLA, EDUARDO MARIA;CACCIOLA, FRANCESCO;DONATO, Paola Agata Eustochia;DUGO, Paola;MONDELLO, Luigi
2012-01-01
Abstract
One of the biggest challenges for scientists in analyzing proteomic samples is related to the presence of thousand of compounds occurring in a wide concentration range. The complexity of such samples becomes even greater after enzymatic digestion to peptides, as a result it is crucial to develop technologies with extremely good resolving power and with extraordinary sensitivity to tackle such a task. Two dimensional capillary and nano liquid chromatography has reduced the requirement for loading large amounts of samples, which is important for the analysis of low-abundance compounds or available in limited quantities. In our approach we developed a comprehensive two-dimensional liquid chromatography (LC×LC) platform comprised of two capillary C18 columns in both dimensions hyphenated by means of two electronically controlled six-port two-positions nano switching valves. Such a system was operated under stop-flow conditions while orthogonality was ensured by the employment of the different pH in both dimensions. A newly designed software was used for data elaboration and visualization of selected peaks in the 2D plane. Good repeatability of retention times and peak areas was attained, holding promise for an application in real-case studies; current research is devoted to improve and speed up analysis by capillary monolithic columns and to optimize MS detection for search into database and protein identification.Pubblicazioni consigliate
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