The results of our analyses demonstrate that our protein microarray-based methodology can reliably and accurately profile PTM conjugation activities in simple (e.g
. purified PTM ligases) and complex (e.g
. whole-cell extracts) biological samples. The assay system is highly reproducible, sensitive (can be performed with as little as 2 µg of whole-cell extract), rapid (analysis can be completed in a single day), and can be easily adapted to profile a variety of different PTM conjugation activities. In this study, we used our assay to 1) identify novel substrates of the SCFSkp2
ubiquitin ligase, 2) profile ubiquitin, SUMO1, and NEDD8 conjugation activities of whole-cell extracts, and 3) define changes in ubiquitylation activity that associate with human breast tumor progression. As further validation of this methodology, during the preparation of this manuscript another group used a similar approach to identify novel substrates of the APC ubiquitin ligase 
Current techniques used to identify substrates of PTMs on a proteome-wide scale include two-hybrid and high-copy suppressor screens in yeast and mass spectrometry 
. However, these techniques have several limitations. For example, PTM analysis by proteomic mass spectrometry can be hindered by 1) low substrate abundance, a characteristic of many ubiquitylated proteins, and/or a sub-stoichiometric level of PTM, 2) the labile nature of many PTMs, making their preservation through biochemical purification, separation, fragmentation, and analysis problematic, especially if native conditions are required leaving substrates vulnerable to de-conjugating enzymes, 3) the adverse effects of certain PTMs on proteases, ionization, and detection efficiency, and 4) multi-site or multi-species modifications, which could make data interpretation problematic.
Our methodology overcomes many of these limitations and provides several advantages over these currently employed techniques. Since our assay relies on the intrinsic PTM conjugation activity of a specimen it is less sensitive to substrate concentrations and sub-stoichiometric modifications can be easily detected. The reactions can also be performed with crude extracts eliminating elaborate purification protocols that could promote de-conjugation of the PTMs. Furthermore, we have successfully multiplexed our assay system to simultaneously profile the conjugation activities of several different PTMs simultaneously on a single protein microarray using differentially labeled fluorescent antibodies for PTM detection (data not shown).
However, there are some potential limitations with our assay system. First, the protein microarrays used in this study display ~8,000 human proteins, representing only ~1/3 of the proteome. Secondly, since the protein microarrays are produced with recombinant human proteins expressed in Sf9 insect cells a proportion of these substrates could be misfolded, possibly precluding their modification or promoting their artificial modification. Thirdly, our methodology may underestimate the number of proteins post-translationally modified if the substrates are printed on the microarrays in a manner that masks a specific sequence that must be recognized by the PTM conjugating enzyme, such as the ubiquitin ligase APC/CCDC20
which uses a destruction box motif (termed D box) for recognition 
. Another potential scenario for this underestimation could be that the arrayed proteins are pre-modified by the conjugation activity in insect cells prior to spotting on the protein microarrays. This may at least hold true for ubiquitylation, since there is evidence that exogenously expressed proteins in Sf9 insect cells can be ubiquitylated in vivo 
. However, evidence suggests that even though they contain SUMOylation machinery, Sf9 cells cannot support SUMOylation of exogenously expressed human proteins 
. Fourthly, being a purely in vitro
assay, in vivo
regulatory processes (e.g.
temporal or spatial regulations) will likely be lost during extract preparation. Finally, information regarding the site of PTM attachment to a substrate cannot be ascertained. Therefore, our assay system might be most effective when it is used in conjunction with other screening techniques and any conjugation activities identified should be thoroughly validated in vivo
Considering that dysfunction of PTMs play a critical role in a number of pathological states in humans, this methodology is an important step forward in the field of proteomics because it will allow for alterations of PTM activities associated with human diseases to be identified. For example, SUMOylation is known to play an important role in maintaining genomic integrity and preventing tumorigenesis. The SUMOylation machinery is recruited to sites of DNA damage, and both the tumor suppressor BRCA1 and the DNA repair factor 53BP1 are substrates of SUMOylation 
. Our methodology could be used to further unravel the role of SUMOylation in the DNA damage repair process, such as through comparison profiling of SUMOylation activities from extracts prepared from UV-irradiated and control cells. A comparison of extracts from normal and cancer cells with defective DNA damage repair might also help to define how this process is dysregulated in cancers. Another example are the deubiquitylating enzymes (DUBs), which function to counteract the E3 ubiquitin ligases by removing ubiquitin from substrates and may play an important role in cancer. One such DUB is A20, which is an NFκB inhibitor and tumor suppressor 
. However, the molecular substrates of A20 are largely unknown. Our methodology might be employed for these studies by incubating protein microarrays that were pre-ubiquitylated by cellular extracts with recombinant A20 protein and profiling for losses in substrate fluorescence.
In combination with genetic mutants, small molecule perturbants, or RNAi technology, our methodology could help to define both specific and global aspects of PTMs. Modified cell lines, disease model systems, and specialized tissues all lend themselves well to PTM profiling using this approach with the ultimate goal of furthering our understanding of disease states and identifying novel therapeutic targets for their treatment.