Prioritization of candidate proteins based on pI and Mw. Step 1: pI and Mw (Da) of the mature forms of the proteins identified by PMF are determined using the ExPASy tool “Compute pI/Mw” . Step 2: The (x,y) coordinates of the identified spots and their corresponding pI and Mw (on log10-scale) are used as training data for fitting two cubic smoothing splines. Step 3: For an unidentified test spot u, a candidate list of proteins is generated using the ExPASy tool TagIdent  by specifying ranges Δ and δ(%) around the pI and Mw predicted by the smoothing splines, respectively. Step 4: Proteins in the candidate list are ranked by calculating their similarities with the PMF-identified ‘seed’ proteins using STRING association scores. Step 5 (optional): The ranked candidate list can be further filtered using presence (black) and absence (white) calls from the Gene Expression Barcode 3.0 . A protein is excluded from the ranked list if the corresponding gene is expressed on none of the selected microarrays.