Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introducing a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products were achieved on an amine-reactive hydrogel coated microarray glass surface, allowing highthroughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in-situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine induced disease free breast cancer patients and in patients, with confirmed disease at time of diagnosis.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

No abstract

O -Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O -glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O -glycans in biological material. While various successful strategies for the in-depth profiling of released O -glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O -glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O -glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O -glycan types and structures, we were able to annotate individual O -glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O -glycan structures, including O -fucose, O -glucose, O -GlcNAc, and O -GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.