Fig. 4

Multi-pronged Pitchfork strategy (A) Pitchfork strategy workflow: all tumor and control (NAM) samples were analyzed using four parallel methods and different ways of sample processing with separate liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. (1) SDC-trypsin: standard proteomic analysis of total sample lysate, (2) N-glyco-FASP: glycopeptide enrichment using lectins, (3) SPEG: glycopeptide enrichment using hydrazide chemistry, and (4) hPTC: enrichment of hydrophobic membrane-embedded segments of IMPs. MS data were searched and proteins identified and quantified (LFQ) for each of the method separately, with the exception of the two glycopeptide “prongs,” where the MS data from both glycocapture methods were searched together. Identified upregulated proteins were filtered for presence of predicted transmembrane segment (TMHMM) and the resulting list of upregulated IMPs was further filtered for cell-surface localization. (B) Upregulated cell-surface IMPs identified in the 22 analyzed PPGLs using the complete Pitchfork workflow (corresponding primary gene names are shown). Proteins identified via standard proteomic analysis are shown in bold letters, proteins identified using glycocapture or hPTC are underlined. Proteins identified using both, total cell lysate analysis and membrane-aimed methods, are indicated by underlined bold letters