Journal: Scientific Reports
Article Title: A misprocessed form of Apolipoprotein A-I is specifically associated with recurrent Focal Segmental Glomerulosclerosis
Figure Lengend Snippet: Urinary ApoA-Ib contains 3 extra amino acids at the N-Terminal end compared to plasmatic mature ApoA-I (form 0). Urine ( A ) and plasma samples ( B ) of ApoA-Ib positive FSGS recurrent patients were resolved in 24-cm 2D SDS-PAGE gels using a 4–7 Ph range and stained with colloidal coomassie. The complete 2DE gels obtained using urine and plasma samples are depicted in panels A and B, respectively. A zoom box of the ApoA-I region detailing the spots analysed in urine (panel A) and in plasma (panel B) is shown. Retinol-binding protein 4 (RET4) is highlighted as a reference spot. The spots corresponding to different forms of ApoA-I were excised, digested with trypsin and run on an LTQ-Orbitrap mass spectrometer. The sequence obtained in each case is shown in bold red and the detected N-Terminal end of each form is marked with an arrow. As can be observed in panel A, ApoA-Ib sequence contained 3 extra aminoacids (WQQ, underlined) at the N-Terminal end that were not present in the ApoA-I form 0.These three amino acids are part of the propeptide sequence that was observed complete in plasma proApoA-I (form +2) (RHFWQQ, underlined) (Panel B). A representative MS/MS scan of the N-terminal peptide of ApoA-Ib can be found in Supplemental Fig. 3 . RET4: Retinol binding protein 4 .
Article Snippet: The tryptic peptides were extracted in a two-step procedure using sonication and were analyzed on an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific) coupled to a nanoAcquity UPLC system (Waters) using a 45 min method.
Techniques: SDS Page, Staining, Two-Dimensional Gel Electrophoresis, Binding Assay, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy