Ge 2d quant kit

The protein sample solution was provided by Impossible Foods Inc. from the LegHb production run PP‐PGM2‐15‐320‐101. The LegHb protein represented 66% of the total protein according to the certificate of analysis. The total protein concentration was determined to be 79.94 mg mL^–1 using a GE 2D Quant kit (GE Healthcare, #80‐6483‐56), following the kit instructions. The PP‐PGM2‐15‐320‐101 is a representative batch of soy LegHb protein preparation. The type and abundance of Pichia proteins is consistent from batch‐to‐batch. Bovine Hb (Sigma–Aldrich Co. LLC., St. Louis, MO, #H2625‐25G), BSA (Sigma‐Aldrich Co. LLC., MO #A9647‐100G), and chicken ovalbumin (OVA; Worthington Biochemicals, #3054) were used as control proteins in the digestion assay.
Pepsin A (Worthington Biochemicals, #3319) with a certificate of analysis of 2810 activity units per mg solid was used in all assays. Novex® 10–20% tris‐glycine polyacrylamide gels (Invitrogen) were used to separate digested materials. Precision Plus Protein^TM dual xtra standards (BioRad) were used as the molecular weight standards. Tris‐Glycine‐SDS 10 × running buffer (Fisher Scientific), Coomassie Brilliant Blue R‐250 staining solution, and Coomassie Brilliant Blue R‐250 destaining solution (BioRad) were used for gel running, staining, and destaining, respectively.

Eight lens samples from four groups were processed and individually examined. Protease inhibitor (Roche Complete ULTRA tablets, mini EASY pack) was added to each sample prior to the experiments. Cold acetone was subsequently added at 4 times the volume of the pellet, and the sample was precipitated at −20 °C overnight. The sample was subsequently centrifuged twice at 14,000 x g for 10 min at 4 °C. Subsequently, the pellet was air-dried, and protein lysis was performed in lysis buffer. The proteins were quantified using the GE 2-D Quant Kit according to the manufacturer’s instructions. Each sample containing 100 μg of quantified protein was enzymolyzed with trypsin and labelled with iTRAQ reagent using the iTRAQ Reagent-8Plex Multiplex Kit (AB SCIEX 4381663) according to the manufacturer’s instructions. The three regenerative lenses were labelled with iTRAQ reagents 113, 114, and 115; the three CC lenses were labelled with iTRAQ reagents 116, 117, and 118; and the ARC and normal lenses were labelled with iTRAQ reagents 119 and 121, respectively. Figure 1 illustrates the workflow of the detailed experiment procedures.Fig. 1

Flowchart of the proteogenomic analysis of the human RLSC, CC and ARC lenses