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A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional activity is not understood. We used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.

Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.

The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Nonrecognition of the presented peptide ultimately leads to cell destruction. Characterizing the factors associated with nonrecognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry. Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I and Class II MHC molecules. The goal of the assay optimization was to minimize the amount of antibody required for the assay, to minimize the amount of biological material needed from which the complex is isolated and to achieve the optimum sensitivity towards the peptides of interest. Citation Format: Michael Ford, Richard Jones, David Allen, Ravi Amunugama, Paul Del Rizzo, Michael Pisano, James Mobley, Paul Domanski, Bill Ho, Daniel Bochar, Melissa Holt. Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B80.

The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Non recognition of the presented peptide ultimately leads to cell destruction. Characterizing the factors associated with Non recognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry. Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I MHC molecules. The goal of the assay optimization was to minimize the amount of antibody required for the assay, to minimize the amount of biological material needed from which the complex is isolated and to achieve the optimum sensitivity towards the hitherto unknown target peptides. Citation Format: Michael Ford, Richard Jones, David Allen, Ravi Amunugama, Paul Del Rizzo, Michael Pisano, James Mobley, Paul Domanski, Bill Ho, Daniel Bochar. Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2017-1673

The macromolecular complex Mediator plays important roles in regulation of RNA Polymerase II (RNAPII) activity by DNA-binding proteins, non-coding RNAs, and chromatin. Structural and biochemical studies have shown that human Mediator exists in two forms; a core complex of 26 proteins, termed Mediator, and a larger complex containing the CDK8 kinase module, termed CDK8-Mediator. Interestingly, 3 subunits of the kinase module have undergone independent gene duplications in vertebrates to generate the paralog pairs MED12/MED12L, MED13/ MED13L, and CDK8/CDK19. Each has been shown to interact with Mediator, yet clearly defining the composition of CDK kinase module has been challenging due to the large size (~600 kD), the similarities between paralogs, and potential combinatorial nature of complexes. In this study, we performed a systematic proteomic analysis using HaloTag technology to isolate each kinase module member (MED12, MED12L, MED13, MED13L, CDK8, CDK19, and Cyclin C) and their interacting partners from HEK293T cells. Using LC-MS/MS we were able to differentiate paralogs within samples by specific analysis of unique peptides. We found that the paralogs assemble into CDKMediator in a mutually exclusive fashion, thus allowing for up to eight different assemblies of the CDK module, with potential for functional specialization of Mediator complexes. Interestingly, we found thatMED13L complexes carry the MED26 subunit, which has been shown to be either absent or in very low abundance in CDK-Mediator. This unique variant of the Mediator complex may reconcile recent observations demonstrating roles for MED26 and CDK8 in positive control of RNAPII elongation. Together these data expand the understanding of CDK-Mediator complexes and composition.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.