Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

Development and validation of an LC-MS/MS method for simultaneous quantification of co-administered trastuzumab and pertuzumab.

Given the increasing use of combination therapy with multiple monoclonal antibodies (mAbs), there is a clinical need for multiplexing assays. For the frequently co-administered anti-human epidermal growth factor receptor 2 (HER2) mAbs trastuzumab and pertuzumab, we developed a high-throughput and robust hybrid ligand-binding liquid chromatography-mass spectrometry (LC-MS)/MS quantitative assay. Nanomolar concentrations of trastuzumab and pertuzumab were determined in 10 µL serum samples after extraction by affinity purification through protein A beads, followed by on-bead reduction, alkylation, and trypsin digestion. After electrospray ionization, quantification was obtained by multiple reaction monitoring LC-MS/MS using SILuMab as an internal standard. The method was validated according to the current guidelines from the US Food and Drug Administration and the European Medicines Agency. Assay linearity was established in the ranges 0.250-250 µg/mL for trastuzumab and 0.500-500 µg/mL for pertuzumab. The method was accurate and selective for the simultaneous determination of trastuzumab and pertuzumab in clinical samples, thereby overcoming the limitation of ligand binding assays that cannot quantify mAbs targeting the same receptor. Furthermore, this method requires a small blood volume, which reduces blood collection time and stress for patients. The assay robustness was verified in a clinical trial where trastuzumab and pertuzumab concentrations were determined in 670 serum samples. As we used commercially available reagents and standards, the described generic bioanalytical strategy can easily be adapted to multiplex quantifications of other mAb combinations in non-clinical and clinical samples.

Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition.

Tumor responses to cancer therapeutics are generally monitored every 2-3 months based on changes in tumor size. Dynamic biomarkers that reflect effective engagement of targeted therapeutics to the targeted pathway, so-called "effect sensors", would fulfill a need for non-invasive, drug-specific indicators of early treatment effect. Using a proteomics approach to identify effect sensors, we demonstrated MUC1 upregulation in response to epidermal growth factor receptor (EGFR)-targeting treatments in breast and lung cancer models. To achieve this, using semi-quantitative mass spectrometry, we found MUC1 to be significantly and durably upregulated in response to erlotinib, an EGFR-targeting treatment. MUC1 upregulation was regulated transcriptionally, involving PI3K-signaling and STAT3. We validated these results in erlotinib-sensitive human breast and non-small lung cancer cell lines. Importantly, erlotinib treatment of mice bearing SUM149 xenografts resulted in increased MUC1 shedding into plasma. Analysis of MUC1 using serial blood sampling may therefore be a new, relatively non-invasive tool to monitor early and drug-specific effects of EGFR-targeting therapeutics.

A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function.

Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP) transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα). Protein lysine acetylation is a key post-translational modification (PTM) that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT) p300 and deacetylated by the lysine deacetylase (KDAC) sirtuin1 (SIRT1). SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+) and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply.

A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis.

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gßγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling.

Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

Protein costs do not explain evolution of metabolic strategies and regulation of ribosomal content: does protein investment explain an anaerobic bacterial Crabtree effect?

Protein investment costs are considered a major driver for the choice of alternative metabolic strategies. We tested this premise in Lactococcus lactis, a bacterium that exhibits a distinct, anaerobic version of the bacterial Crabtree/Warburg effect; with increasing growth rates it shifts from a high yield metabolic mode [mixed-acid fermentation; 3 adenosine triphosphate (ATP) per glucose] to a low yield metabolic mode (homolactic fermentation; 2 ATP per glucose). We studied growth rate-dependent relative transcription and protein ratios, enzyme activities, and fluxes of L. lactis in glucose-limited chemostats, providing a high-quality and comprehensive data set. A three- to fourfold higher growth rate rerouted metabolism from acetate to lactate as the main fermentation product. However, we observed hardly any changes in transcription, protein levels and enzyme activities. Even levels of ribosomal proteins, constituting a major investment in cellular machinery, changed only slightly. Thus, contrary to the original hypothesis, central metabolism in this organism appears to be hardly regulated at the level of gene expression, but rather at the metabolic level. We conclude that L. lactis is either poorly adapted to growth at low and constant glucose concentrations, or that protein costs play a less important role in fitness than hitherto assumed.

Susceptibility to COPD: differential proteomic profiling after acute smoking.

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD.

Lactococcus lactis†YfiA is necessary and sufficient for ribosome dimerization.

Dimerization and inactivation of ribosomes in Escherichia coli is a two-step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactis MG1363 expresses a protein, YfiA(L) (l) , which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full-length YfiA(L) (l) is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy-starving conditions. The N-terminal domain of YfiA(L) (l) is homologous to HPF from E. coli, whereas the C-terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N- and C-terminal domains of YfiA(L) (l) . It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two-dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli.

Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii.

LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the core of Photosystem II (PSII) to form multiprotein complexes. Several PSII supercomplexes with different antenna sizes have been purified, the largest of which contains three LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13Å resolution was obtained allowing the reconstruction of the 3D structure of the supercomplex. The position and orientation of the S trimer are the same as in plants; trimer M is rotated by 45° and the additional trimer (named here as LHCII-N), which is taking the position occupied in plants by CP24, is directly associated with the core. The analysis of supercomplexes with different antenna sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the trimers in the PSII supercomplex, while LhcbM5 is part of the "extra" LHCII pool not directly associated with the supercomplex. It is also shown that Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex from plants and a blue shifted absorption spectrum. Finally the data indicate that there are at least six LHCII trimers per dimeric core in the thylakoid membranes, meaning that the antenna size of PSII of C. reinhardtii is larger than that of plants.

Proteolysin, a novel highly thermostable and cosolvent-compatible protease from the thermophilic bacterium Coprothermobacter proteolyticus.

Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.

Proteomic analysis of human epithelial lining fluid by microfluidics-based nanoLC-MS/MS: a feasibility study.

Microfluidics-based nanoLC-MS/MS (chipLC-MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D-PAGE followed by in-gel tryptic digestion and chipLC-MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non-COPD controls using chemical stable isotope labeling (iTRAQ®-8Plex) showed that the levels of lactotransferrin, high-mobility group protein B1 (HMGB 1), alpha 1-antichymotrypsin and cofilin-1 differed significantly in ELF from COPD patients and non-COPD controls (p-values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non-COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC-MS/MS and quantitative proteomics in human ELF.

Dictyostelium Ric8 is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis.

Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the complex into Gα-GTP and a Gßγ dimer. In Dictyostelium, a basal chemotaxis pathway consisting of heterotrimeric and monomeric G proteins is sufficient for chemotaxis. Symmetry breaking and amplification of chemoattractant sensing occurs between heterotrimeric G protein signaling and Ras activation. In a pull-down screen coupled to mass spectrometry, with Gα proteins as bait, we have identified resistant to inhibitors of cholinesterase 8 (Ric8) as a nonreceptor guanine nucleotide exchange factor for Gα-protein. Ric8 is not essential for the initial activation of heterotrimeric G proteins or Ras by uniform chemoattractant; however, it amplifies Gα signaling, which is essential for Ras-mediated symmetry breaking during chemotaxis and development.

GxcC connects Rap and Rac signaling during Dictyostelium development.

BACKGROUND: Rap proteins belong to the Ras family of small G-proteins. Dictyostelium RapA is essential and implicated in processes throughout the life cycle. In early development and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K and it regulates substrate attachment and myosin disassembly via the serine/threonine kinase Phg2. RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process. RESULTS: Here we show that cells expressing constitutively active RapA exhibit a high level of Rac activation. With a pull-down screen coupled to mass spectrometry, we identified the Rac specific guanine nucleotide exchange factor, GxcC, as Rap binding partner. GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins. Deletion studies revealed that this pathway is involved in regulating Dictyostelium development. CONCLUSIONS: GxcC provides a novel link between Rap and Rac signalling and is one of the Rap effectors regulating the progression of multicellular development.

Nonredundant and locus-specific gene repression functions of PRC1 paralog family members in human hematopoietic stem/progenitor cells.

The Polycomb group (PcG) protein BMI1 is a key factor in regulating hematopoietic stem cell (HSC) and leukemic stem cell self-renewal and functions in the context of the Polycomb repressive complex 1 (PRC1). In humans, each of the 5 subunits of PRC1 has paralog family members of which many reside in PRC1 complexes, likely in a mutually exclusive manner, pointing toward a previously unanticipated complexity of Polycomb-mediated silencing. We used an RNA interference screening approach to test the functionality of these paralogs in human hematopoiesis. Our data demonstrate a lack of redundancy between various paralog family members, suggestive of functional diversification between PcG proteins. By using an in vivo biotinylation tagging approach followed by liquid chromatography-tandem mass spectrometry to identify PcG interaction partners, we confirmed the existence of multiple specific PRC1 complexes. We find that CBX2 is a nonredundant CBX paralog vital for HSC and progenitor function that directly regulates the expression of the cyclin-dependent kinase inhibitor p21, independently of BMI1 that dominantly controls expression of the INK4A/ARF locus. Taken together, our data show that different PRC1 paralog family members have nonredundant and locus-specific gene regulatory activities that are essential for human hematopoiesis.

Impact of Lactobacillus plantarum sortase on target protein sorting, gastrointestinal persistence, and host immune response modulation.

Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L. plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiCl protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation.

A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.

Interactions between hematopoietic stem cells and their niche are mediated by proteins within the plasma membrane (PM) and changes in these interactions might alter hematopoietic stem cell fate and ultimately result in acute myeloid leukemia (AML). Here, using nano-LC/MS/MS, we set out to analyze the PM profile of two leukemia patient samples. We identified 867 and 610 unique CD34(+) PM (-associated) proteins in these AML samples respectively, including previously described proteins such as CD47, CD44, CD135, CD96, and ITGA5, but also novel ones like CD82, CD97, CD99, PTH2R, ESAM, MET, and ITGA6. Further validation by flow cytometry and functional studies indicated that long-term self-renewing leukemic stem cells reside within the CD34(+)/ITGA6(+) fraction, at least in a subset of AML cases. Furthermore, we combined proteomics with transcriptomics approaches using a large panel of AML CD34(+) (n = 60) and normal bone marrow CD34(+) (n = 40) samples. Thus, we identified eight subgroups of AML patients based on their specific PM expression profile. GSEA analysis revealed that these eight subgroups are enriched for specific cellular processes.

Effect of iTRAQ labeling on the relative abundance of peptide fragment ions produced by MALDI-MS/MS.

The identification of proteins in proteomics experiments is usually based on mass information derived from tandem mass spectrometry data. To improve the performance of the identification algorithms, additional information available in the fragment peak intensity patterns has been shown to be useful. In this study, we consider the effect of iTRAQ labeling on the fragment peak intensity patterns of singly charged peptides from MALDI tandem MS data. The presence of an iTRAQ-modified basic group on the N-terminus leads to a more pronounced set of b-ion peaks and distinct changes in the abundance of specific peptide types. We performed a simple intensity prediction by using a decision-tree machine learning approach and were able to show that the relative ion abundance in a spectrum can be correctly predicted and distinguished from closely related sequences. This information will be useful for the development of improved method-specific intensity-based protein identification algorithms.

Regulation of ykrL (htpX) by Rok and YkrK, a novel type of regulator in Bacillus subtilis.

Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, and salt and phenol stress) point to the involvement of YkrL in membrane protein quality control.