Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock-induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and ␤-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and ␤-hydroxypropanoic acidmodified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first suc-

Identification of the molecular targets for post-translational modifications is an important step for explaining the regulated pathways. The ubiquitin-like molecule NEDD8 is implicated in the regulation of cell proliferation, viability and development. By combining proteomics and in vivo NEDDylation assays, we identified a subset of ribosomal proteins as novel targets for the NEDD8 pathway. We further show that the lack of NEDDylation in cells causes ribosomal protein instability. Our studies identify a novel and specific role of the NEDD8 pathway in protecting a subset of ribosomal proteins from destabilization.

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