FD-LC-MS/MS Method for Determining Protein Expression and Elucidating Biochemical Events in Tissues and Cells

Abstract

To study biochemical events in tissues and cells, we have developed a novel proteomics approach, FD-LC-MS/MS, which consists of fluorogenic derivatization (FD), LC separation and detection/quantification of proteins in a biological sample, followed by the isolation and tryptic digestion of target proteins, and then their identification using nano-HPLC-MS/MS. Fluorogenic reagents such as 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulphonamide (DAABD-Cl) were designed to have high sensitivity for HPLC-fluorescence and -MS/MS detection and reactivity for cysteine residues in proteins. This comprehensive differential proteomics approach was applied to several tissues, such as mouse liver, mouse brain, horse muscle, breast cancer cell lines, and mouse heart, in order to study fluctuations in protein levels in tissues and cells.

Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed‐phase liquid chromatographic system

“…However, it has generally been difficult to separate mixtures containing thousands of proteins with sufficient resolution in a single HPLC run. We have chosen HPLC to separate highly complex mixtures of intact proteins and developed an original proteomics method, called fluorogenic derivatized (FD) liquid chromatography–tandem mass spectrometry (FD‐LC‐MS/MS; Imai et al ., ; Ichibangase and Imai, ; Koshiyama et al ., ). Quantification and identification of proteins with this method involve several steps: first, the protein mixtures in bio‐samples are directly derivatized at cysteine residues with a fluorogenic reagent, such as 7‐chloro‐ N ‐[2‐(dimethylamino) ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide (DAABD‐Cl).…”

“…The FD‐LC‐MS/MS method has been applied to real bio‐samples, such as extracts from mouse liver (Ichibangase et al ., ), mouse brain (Asamoto et al ., ), horse muscle (Ichibangase and Imai, ), breast cancer cell lines (Imai et al ., ), rat kidney (Tsai et al ., ), colorectal cancer cell lines (Koshiyama et al ., ) and superoxide dismutase 1 knockout cells (Ichibangase et al ., ), and has been used to elucidate intracellular biochemical events. Meanwhile, the separation conditions for further high‐performance separation of intact proteins derivatized with DAABD‐Cl have been optimized as far as the operational limits of conventional HPLC will allow (Masuda et al ., ; Ichibangase et al ., , ; Ichibangase and Imai, ). With regard to the HPLC column, the use of nonporous reversed‐phase materials (i.e.…”

“…Josic and Kovac () reported that elevated temperature enhanced kinetic and transport properties during protein chromatographic separation, while others have reported that flow rate, separation time, eluent pH, the presence of ion‐pairing reagents in the eluent and column length all potentially influence intact protein separation (Josic and Kovac, ; Lestremau et al ., ; Yang et al ., ; Lasaosa et al ., ). However, relatively little work has been carried out on the effects of variables on the chromatographic separation of high‐molecular‐weight compounds and of highly complex protein mixtures, and there are no groups other than our own that perform the separation of intact protein mixtures at the level of hundreds of peaks (Ichibangase and Imai, ).…”

Proteomics analysis of altered proteins in kidney of mice with aristolochic acid nephropathy using the fluorogenic derivatization–liquid chromatography–tandem mass spectrometry method

“…A fluorogenic derivatization–liquid chromatography–tandem mass spectrometry (FD‐LC–MS/MS) method has been successfully established to separate proteins (Masuda, Toriumi, Santa, & Imai, ; Toriumi & Imai, ). The FD‐LC–MS/MS method, which is capable of analyzing comprehensively the expressed proteins, has been widely applied to proteomics analysis at various scales from cells to mammalian tissues (Asamoto, Ichibangase, Uchikura, & Imai, ; Ichibangase et al, ; Ichibangase & Imai, ; Ichibangase, Moriya, Koike, & Imai, ; Koshiyama, Ichibangase, & Imai, ; Masuda, Saimaru, Takamura, & Imai, ; Tsai et al, ). This FD‐LC–MS/MS method, which is much rapid, effective and more sensitive than the traditional 2D–PAGE method used for proteomics study, consists of the following steps: derivatization of protein using the fluorogenic agent 7‐chloro‐ N ‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide (DAABD‐Cl); separation and quantification of derivatized proteins by high‐performance liquid chromatography (HPLC); identification by FD‐LC–MS/MS with the MASCOT database searching system (Imai et al, ; Masuda et al, ). …”

Progress and Clinical Applications in Proteomics

“…Proteomics research has investigated the entire protein content of various organisms; in other words, it is functional genomics at the level of proteins. Proteomics-based approaches have used human plasma [7], serum [8], urine [9], cerebrospinal fluid [10], nipple aspirate fluid [11], ductal lavage [12], amniotic fluid [13], bile [14], lymph [15], breast milk [16], mucus [17], pleural fluid [18], saliva [19], tears [20], and various tissues and cells [21] as protein sources. Thus, the clinical application of this approach (clinical proteomics) can be applied to identify specific disease markers, biomarkers, drug and therapeutic targets.…”