Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library

The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase–substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library–kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in <i>Escherichia coli</i>, we perform <i>in vitro</i> kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCβ mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future

Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library

The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase–substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library–kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in <i>Escherichia coli</i>, we perform <i>in vitro</i> kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCβ mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future

Comparative Proteomics Enables Identification of Nonannotated Cold Shock Proteins in <i>E. coli</i>

Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions