Rapid and deep-scale ubiquitylation profiling for biology and translational research

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples

Large-scale identification of ubiquitination sites by mass spectrometry.

Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of antibodies that recognize the Lys-{epsilon}-Gly-Gly (K-{epsilon}-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-{epsilon}-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC-tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ~5 d

Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-{epsilon}-GG) antibodies. Here, we describe a number of improvements to the K-{epsilon}-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ~20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input

iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ(TM) or TMT(TM) allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ(TM) yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification

Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition

Ubiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-{epsilon}-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K-ε-GG peptides three- to fourfold resulting in detection of up to ~3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K-{epsilon}-GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by P-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study

Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics

Integrated proteomic analysis of post-translational modifications by serial enrichment

We report a mass spectrometry-based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells

HOLDING BACK THE TEARS! An autoethnographic study of the struggles of a hospital teacher in balancing emotions, care and empathy

The purpose of this action research self-study was to address the challenging tensions I experienced as I embarked on a new stage of my career as a teacher in a hospital school setting. My overarching aim was to use the insights gleaned to become a more effective hospital teacher. Autoethnography as a methodology, revealed that my tensions were concerned with three integrated areas: emotions, my ontological value of care, and empathy. Upon discovering this, I refined the overarching question for the study to; as a hospital teacher, how can I balance tensions of teaching and caring for students in an emotionally challenging environment? The rationale behind this research approach stemmed from ethical considerations and the professional and personal challenges I encountered, following the move from mainstream teaching to hospital teaching. My practices, beliefs and values were questioned by the experiences I faced in this unusual educational setting. A qualitative research design was chosen and autoethnography was selected as a methodology as a natural consequent to the aims of the study. To achieve these aims, it required an introspective research design, which valued ‘self’ as the researched. It also acknowledges vulnerabilities and emotions as valuable personal data. Significantly, students and parents were considered an extremely vulnerable cohort in this particular educational context. Therefore, I made the ethically informed decision to exclude this cohort from my research. Autoethnography facilitates their absence without damaging the richness and value of the study. As mentioned, autoethnography relies on personal data, of which the following were utilised in this study: personal memory, archived data, reflections, reflective writing, journal, metaphor, photographs, and poetry. Validity and rigour were established by the ‘outsider’ perspective gained from relevant literature, my critical friend, my supervisor and interviews with four teacher participants. This study found that emotional challenges are the greatest challenges faced in the educational setting of the hospital school. Personal responsibility is required to identify and enhance emotional intelligence competencies, specifically my self-awareness, selfregulation and empathy. The latter has a tripartite classification, with emotional well-being being negatively affected by the practice of emotional empathy, as opposed to a more favourable form—compassionate empathy. My own supressed grief and the fight to hold back my emotions was unearthed during my writing. The hospital context brought to the surface supressed grief and emotionality, my empathy for others, and my desire to care. Finally, I determined that my value of care was grounded in characteristics of empathy and maternal traits. Reflection on data retrieved from the ‘outsider’ resulted in my reconceptualization of care as multi-dimensional, and categorised under students, practice and self. I can conclude from this study that I need to engage with self-care. This will involve enhancing my emotional intelligence competencies. Additionally, I need to practice emotional labour at work and deal with the emotional experiences appropriately and timely, in the correct environment. Lastly, my identity as a hospital teacher has been enhanced. The confident mainstream teacher that I once was is beginning to re-emerge in this new and challenging setting. This is due to my renewed self-awareness of my emotionality, my understanding of empathy, and reconceptualization of my core value, care. I now go about my work with a newfound sense of confidence and pride. The cumulative learning gained from this study is life enhancing and life changing. I am now a hospital teacher who can articulate clearly and enact in my practice what it means to balance education and care

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry

Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from 0.88. The maximum deviation for the phosphoproteome coverage was 37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration