PGNMID Proteome

Human renal biopsy samples from Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposits (PGNMID) patinets were obtained. Renal glomeruli were laser microdissected and subjected to shotgun proteomics analysis. Raw data files were uploaded. Below is the mass spectrometry protocol used to acquire the data. 

Proteomics Analysis of Renal Glomeruli. Sample cases were processed, beginning with 10-uM thick formalin-fixed paraffin sections (FFPE) that were mounted on PEN membrane glass slides then deparaffinized in xylene and rehydrated. The glomeruli were microdissected using a Zeiss Palm Microbeam microscope to accumulate approximately 250000-550000 uM2 per case into the cap of a 0.5ml tube containing 35ul of 100mM Tris pH 8.2 / 0.005% zwittergent 3-16. The collected FFPE fragments were centrifuged and heated at 98°C for 60minutes for protein extraction, then reduced with tris carboxyethyl phosphine (TCEP), alkylated with iodoacetamide followed by digestion with trypsin/LysC mix (Promega) at 37°C overnight. The digest mixture was acidified with dilute trifluoroacetic acid and analyzed by nanoLC-tandem mass spectrometry using a Thermo Scientific Orbitrap Eclipse mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to a Vanguish Neo UHPLC system with the A solvent as 0.1% formic acid in 98% water / 2% acetonitrile and the B solvent as 0.1% formic acid in 80% acetonitrile / 10% isopropanol / 10% water.  The peptide digests were loaded onto a PepMap Neo C18, 5µm, 300µm x 5mm trap with 0.1% formic acid / 0.05%TFA at a flow rate of 8ml / minute.  The trap was placed in line with a 100um x 40cm C18, 2.4µm PepSep column and the peptides separated at a flow rate of 350nl/minute with a gradient of 5%B to 40%B over 90minutes followed with a 20-minute ramp to 95%B and hold for 10 minutes. The Eclipse was set for data dependent acquisition with a 3 second cycle time. The MS1 survey scan range was 340-1600 m/z at resolution 120,000 (@ 200m/z) with the automatic gain control (AGC) set to allow up to 8x105 ions (200%) and the maximum ion inject time set to auto.  Precursor ions in the scan range of 340-1400 m/z, with positive charge states from 2-5, were sequentially selected with an isolation window of 0.7 m/z and fragmented by high energy collisional dissociation (HCD) with a NCE setting of 28%.  The MS/MS scans were acquired at resolution 15,000 with the AGC setting at 100% (5x104 ions) and the max ion inject time set to 50ms.  The dynamic exclusion feature was used to prevent any ion within a 14ppm window of the selected ion, from being chosen again for MS/MS fragmentation for at least 45 seconds.