Proteomes are significantly more complex than genomes and transcriptomes due to protein processing and extensive post-translational modification (PTM) of proteins. Hundreds of different modifications exist. Release 66 of the RESID database(1) (http://www.ebi.ac.uk/RESID/) contains 559 different modifications, including small chemical modifications such as phosphorylation, acetylation, and methylation and modification by small proteins, including ubiquitin and ubiquitin-like (UBL) proteins that are covalently coupled to proteins to regulate their activity. A wide variety of cellular processes are regulated by these reversible modifications, including transcription, replication, cell-cycle progression, and responses to DNA damage. Protein modifications have been studied for many years at the level of single target proteins, but currently available technologies enable proteome-wide studies of these modifications by mass spectrometry (MS).2,3 Powerful proteomics tools are available to study phosphorylation and acetylation at a systems-wide level in a site-specific manner. It is more challenging to study ubiquitin targets and targets for ubiquitin-like proteins at a proteome-wide level in a site-specific manner due to the relatively large size of these modifications, but hundreds of potential target proteins have been uncovered over the past eight years, mainly in a non-site-specific manner. This review is focused on uncovering signaling networks for ubiquitin and ubiquitin-like proteins by mass spectrometry and highlights the site-specific studies published in 2010 and 2011. Site-specific methodologies will likely have a major impact on the ubiquitin field in the near future. The methodology, results, challenges, pitfalls, crosstalk with other PTMs, and future directions are discussed in this review. 1.1. Ubiquitin and Ubiquitin-like Proteins Ubiquitin was first discovered in the mid-1970s, and the 2004 Nobel Prize in Chemistry was awarded for this finding. Ubiquitin is a 76 amino acid protein that is highly conserved from yeast to plants and mammals. Many ubiquitin-like proteins have been uncovered, including Nedd8, small ubiquitin-like modifier 1 (SUMO-1), SUMO-2, SUMO-3, FUBI, HUB1, ISG15, FAT10, URM1, UFM1, Atg12, and Atg8. Ubiquitin-like proteins are also found in prokaryotes and archaea; PUPs are prokaryotic ubiquitin-like proteins, and SAMPs are ubiquitin-like small archaeal modifier proteins. Despite limited sequence homology of some family members with ubiquitin, all ubiquitin family members display structural homology via the characteristic β-grasp ubiquitin fold.4−9 These small proteins are covalently coupled to target proteins via isopeptide bonds between C-terminal diglycine motifs and e-amino groups in lysines of target proteins using an enzymatic cascade that consists of an E1 enzyme,(10) an activator of ubiquitin and UBLs, an E2 enzyme,11,12 and a ligase, known as an E3 enzyme(13) (Figure ​(Figure1).1). Humans express 8 E1 enzymes(10) (including 1 dedicated to ubiquitin, 1 shared between ubiquitin and the UBL FAT10, and 6 dedicated to other UBLs) and 35 active E2 enzymes (including 28 dedicated to ubiquitin, 3 shared between ubiquitin and the UBL ISG15, 3 dedicated to other UBLs, and 1 putative E2).(12) Ubiquitin E3 enzymes are subdivided into HECT-type E3 enzymes (homology to E6AP carboxyl terminus)(14) and RING-type E3 enzymes (really interesting new gene).(15) HECT-type E3 enzymes form thioesters with ubiquitin, whereas RING-type E3 enzymes lack catalytic cysteines. Over 600 human genes encode components of RING-based E3 ligases.(15) Figure 1 Ubiquitylation cascade. Ubiquitin precursors are processed by proteases to generate mature ubiquitin containing a C-terminal diglycine motif for conjugation to target proteins. Three different classes of enzymes are involved: E1, E2, and E3 enzymes. Ubiquitin ...