Fig. 1.

Overview of different protocols for barley leaf protein extraction and digestion. a Flow chart of evaluation of different sample preparation protocols. b Feature summary of different sample preparation protocols. Barley leaf proteins were extracted with three different buffers containing SDS and DTT, SDC and DTT, and SDC, tris(2-carboxyethyl)phosphine (TCEP) and chloroacetamide (CAA), respectively. Proteins extracted with SDS- and DDT-based buffer were transferred to a spin filter for cleaning up with urea and then digested (FASP) or precipitated by TCA/acetone and then subjected to standard in-solution digestion (SDS-ISD). Proteins extracted with SDC- and DTT-based buffer were transferred to a spin filter for cleaning up with SDC and then digested (SDC-FASP) or directly subjected to standard in solution digestion (SDC-ISD). Protein extracted with SDC-, TCEP- and CAA-based buffer were subjected directly to standard in-solution digestion (OP-ISD). SDC used in SDC-FASP, SDC-ISD and OP-ISD protocols was removed by the phase transfer method before mass spectrometer analysis