The conversion of arginine to citrulline in proteins is catalyzed by protein arginine deiminases (PADs). In humans, there are five PAD isozymes with different tissue and subcellular expressions, resulting in different access to substrates. Although it is known that PAD enzymes differ in their protein target spectrum, the influence of substrate sequence motif and its origin are not yet elucidated. To study the motif preferred by the different PAD enzymes, cell lysates were subjected to citrullination by PAD1-4 in vitro, digested with trypsin, fractionated and measured by LC-MS/MS to obtain a deep and comprehensive survey of the citrullination landscape. Using this setup, an average of over 90,000 peptides (~6100 proteins) could be quantified per sample. The extent of citrullination achieved by PAD1-4 ranged between 6-23%, with 8,000-22,000 individual citrullination sites confidently identified across triplicates. Notably, the number of sites generated by PAD1 and 2 was almost double than that of PAD3 and 4. Motif analysis of the citrullinated peptides revealed a distinct preference of both PAD3 and 4 towards D in -1 and D, E, G, and N in +1 position relative to the modified R. PAD1 and 2 showed a more permissive motif, favoring K, D, E and I in the entire region around the citrullination site. To further investigate this difference in substrate preference, we performed site-directed mutagenesis on PAD4, targeting five residues (Q346, R374, G403, R639 and H640) around the substrate binding sites that differ between PAD2 and PAD4. Most of the mutants showed elevated catalytic activity compared to the wild type and increasing similarity in their substrate motif to that of PAD2. Specifically, Q346, R639, and H640 residues were found to play important roles in the substrate selection of PAD4. This work presents the first systematic investigation of motif preferences of PAD1-4 enzymes in the human proteome and identifies key residues involved in substrate selection in PAD4