RAMZIS: a bioinformatic toolkit for rigorous assessment of the alterations to glycoprotein composition that occur during biological processes.

Motivation: Glycosylation elaborates the structures and functions of glycoproteins; glycoproteins are common post-translationally modified proteins and are heterogeneous and non-deterministically synthesized as an evolutionarily driven mechanism that elaborates the functions of glycosylated gene products. Glycoproteins, accounting for approximately half of all proteins, require specialized proteomics data analysis methods due to micro- and macro-heterogeneities as a given glycosite can be divided into several glycosylated forms, each of which must be quantified. Sampling of heterogeneous glycopeptides is limited by mass spectrometer speed and sensitivity, resulting in missing values. In conjunction with the low sample size inherent to glycoproteomics, a specialized toolset is needed to determine if observed changes in glycopeptide abundances are biologically significant or due to data quality limitations. Results: We developed an R package, Relative Assessment of m/z Identifications by Similarity (RAMZIS), that uses similarity metrics to guide researchers to a more rigorous interpretation of glycoproteomics data. RAMZIS uses a permutation test to generate contextual similarity, which assesses the quality of mass spectral data and outputs a graphical demonstration of the likelihood of finding biologically significant differences in glycosylation abundance datasets. Investigators can assess dataset quality, holistically differentiate glycosites, and identify which glycopeptides are responsible for glycosylation pattern change. RAMZIS is validated by theoretical cases and a proof-of-concept application. RAMZIS enables comparison between datasets too stochastic, small, or sparse for interpolation while acknowledging these issues in its assessment. Using this tool, researchers will be able to rigorously define the role of glycosylation and the changes that occur during biological processes. Availability and implementation: https://github.com/WillHackett22/RAMZIS.

Retinal small vessel narrowing in women with gestational diabetes, pregnancy-associated hypertension, or small-for-gestational age babies.

Background: Gestational diabetes, pregnancy-associated hypertension and small-for-gestational age babies are all associated with impaired placental vascularisation. This study compared the effects of these conditions the systemic small vessel calibre that was examined in the retina. Methods: This was a cross-sectional observational study of consecutive pregnant women recruited from an antenatal clinic. Participants underwent a Glucose Tolerance Test, BP measurements, and were examined for small-for-gestational age babies as per national guidelines. They also underwent retinal photography with a non-mydriatic camera, and vessel calibres were measured with a validated semi-quantitative system at a retinal grading centre. Some participants also underwent testing of retinal vascular responsiveness to a flickering light. Results: Women with gestational diabetes (n = 68) had a higher mean arterial pressure (85 ± 9 mm Hg) than normal pregnant women (n = 27, 80 ± 8 mmHg, p = 0.01). They also had smaller mean retinal arteriole (147.5 ± 13.6 µm and 159.7 ± 6.7 µm respectively, p < 0.01) and venular calibre (221.0 ± 13.4 µm and 232.8 ± 20.1 µm respectively, p < 0.01) than normal. However their babies' mean birth weights were not different from normal (3,311 ± 558 g and 3,401 ± 600 g respectively, p = 0.48). They also demonstrated a trend to reduced retinal arteriolar dilatation (3.5 ± 1.3%, n = 23) in response to vasodilatory stimuli (4.4 ± 1.8%) (n = 11) (p = 0.08) consistent with endothelial dysfunction. Women with pregnancy-associated hypertension (n = 35) had a higher mean arterial pressure (101 ± 12 mm Hg, p < 0.01), a smaller mean retinal arteriolar calibre (139.9 ± 10.6 µm, p < 0.0001), and a lower baby mean birth weight than for normal pregnancies (3,095 ± 443 g, p = 0.02). Likewise, women with small-for-gestational age babies (n = 31) had a higher mean arterial pressure (89 ± 19 mm Hg, p = 0.03), a smaller mean retinal arteriolar calibre (141.6 ± 12.8 µm, p < 0.01) and a lower baby mean birth weight than for normal pregnancies (2,468 ± 324 g, p < 0.0001). Conclusion: Mean retinal arterial calibre was reduced in women with gestational diabetes, pregnancy-associated hypertension or small-for-gestational age babies. The reduction in calibre was greatest in pregnancy-associated hypertension and small-for-gestational age babies. Systemic arteriole narrowing may contribute to the pathogenesis of placental vascular dysfunction in these conditions.

RAMZIS: a bioinformatic toolkit for rigorous assessment of the alterations to glycoprotein structure that occur during biological processes.

Motivation: Glycosylation elaborates the structures and functions of glycoproteins; glycoproteins are common post-translationally modified proteins and are heterogeneous and non-deterministically syn-thesized as an evolutionarily driven mechanism that elaborates the functions of glycosylated gene products. While glycoproteins account for approximately half of all proteins, their macro- and micro-heterogeneity requires specialized proteomics data analysis methods as a given glycosite can be divided into several glycosylated forms, each of which must be quantified. Sampling of heterogeneous glycopeptides is limited by mass spectrometer speed and sensitivity, resulting in missing values. In conjunction with the low sample size inherent to glycoproteomics, this necessitated specialized statistical metrics to identify if observed changes in glycopeptide abundances are biologically significant or due to data quality limitations. Results: We developed an R package, Relative Assessment of m/z Identifications by Similarity (RAMZIS), that uses similarity metrics to guide biomedical researchers to a more rigorous interpretation of glycoproteomics data. RAMZIS uses contextual similarity to assess the quality of mass spectral data and generates graphical output that demonstrates the likelihood of finding biologically significant differences in glycosylation abundance dataset. Investigators can assess dataset quality, holistically differentiate glycosites, and identify which glycopeptides are responsible for glycosylation pattern expression change. Herein RAMZIS approach is validated by theoretical cases and by a proof-of-concept application. RAMZIS enables comparison between datasets too stochastic, small, or sparse for interpolation while acknowledging these issues in its assessment. Using our tool, researchers will be able to rigor-ously define the role of glycosylation and the changes that occur during biological processes.

Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry.

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin-Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution.

To teach or not to teach? Assessing medical school faculty motivation to teach in the era of curriculum reform.

BACKGROUND: Medical schools have undergone a period of continual curricular change in recent years, particularly with regard to pre-clinical education. While these changes have many benefits for students, the impact on faculty is less clear. METHODS: In this study, faculty motivation to teach in the pre-clinical medical curriculum was examined using self-determination theory (SDT) as a framework. Basic science and clinical faculty were surveyed on factors impacting their motivation to teach using validated scales of motivation as well as open-ended questions which were coded using self-determination theory (SDT) as a guiding framework. RESULTS: Faculty reported that teaching activities often meet their basic psychological needs of competence, autonomy, and relatedness. Professors were more likely than associate professors to report that teaching met their need for autonomy. Faculty were more motivated by intrinsic as compared to external factors, although basic science faculty were more likely than clinical faculty to be motivated by external factors. Motivating and de-motivating factors fell into the themes Resources, Recognition and Rewards, Student Factors, Self-Efficacy, Curriculum, Contribution, and Enjoyment. The majority of factors tied to the faculty's need for relatedness. Based on these findings, a conceptual model for understanding medical school faculty motivation to teach was developed. CONCLUSIONS: Assessing faculty motivation to teach provided valuable insights into how faculty relate to their teaching roles and what factors influence them to continue in those roles. This information may be useful in guiding future faculty development and research efforts.

I don't think that means what you think it means: Why precision in lifelong learning terminology matters to medical education.

ISSUE: Medical educators share the belief that fostering the development of lifelong learning skills is a fundamental task for teachers and learners in all stages of a physician's education: undergraduate medical education, graduate medical education, and continuing medical education. A significant challenge to developing and implementing best practices in lifelong learning is the varied interpretation and application of its related terminology, such as 'self-directed learning' in this context. EVIDENCE: This paper discusses the scholarly origins of key terms in lifelong learning ('self-directed learning' and 'self-regulated learning') and explores their commonalities and their common conflation. IMPLICATION: The authors propose a renewed attention to precision in use of lifelong learning terminology in medical education across the spectrum as a way to best design and deploy impactful educational experiences for learners at all levels.

The minimum information required for a glycomics experiment (MIRAGE): reporting guidelines for capillary electrophoresis.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. These guidelines are linked to other MIRAGE guidelines and are freely available through the project website https://www.beilstein-institut.de/en/projects/mirage/guidelines/#ce_analysis (doi:10.3762/mirage.7).

Methods to improve quantitative glycoprotein coverage from bottom-up LC-MS data.

Advances in mass spectrometry instrumentation, methods development, and bioinformatics have greatly improved the ease and accuracy of site-specific, quantitative glycoproteomics analysis. Data-dependent acquisition is the most popular method for identification and quantification of glycopeptides; however, complete coverage of glycosylation site glycoforms remains elusive with this method. Targeted acquisition methods improve the precision and accuracy of quantification, but at the cost of throughput and discoverability. Data-independent acquisition (DIA) holds great promise for more complete and highly quantitative site-specific glycoproteomics analysis, while maintaining the ability to discover novel glycopeptides without prior knowledge. We review additional features that can be used to increase selectivity and coverage to the DIA workflow: retention time modeling, which would simplify the interpretation of complex tandem mass spectra, and ion mobility separation, which would maximize the sampling of all precursors at a giving chromatographic retention time. The instrumentation and bioinformatics to incorporate these features into glycoproteomics analysis exist. These improvements in quantitative, site-specific analysis will enable researchers to assess glycosylation similarity in related biological systems, answering new questions about the interplay between glycosylation state and biological function.

Avelumab Combined with Stereotactic Ablative Body Radiotherapy in Metastatic Castration-resistant Prostate Cancer: The Phase 2 ICE-PAC Clinical Trial.

BACKGROUND: Immune checkpoint inhibitor monotherapy in metastatic castration-resistant prostate cancer (mCRPC) has produced modest results. High-dose radiotherapy may be synergistic with checkpoint inhibitors. OBJECTIVE: To evaluate the efficacy and safety of the PD-L1 inhibitor avelumab with stereotactic ablative body radiotherapy (SABR) in mCRPC. DESIGN, SETTING, AND PARTICIPANTS: From November 2017 to July 2019, this prospective phase 2 study enrolled 31 men with progressive mCRPC after at least one prior androgen receptor-directed therapy. Median follow-up was 18.0 mo. INTERVENTION: Avelumab 10 mg/kg intravenously every 2 wk for 24 wk (12 cycles). A single fraction of SABR (20 Gy) was administered to one or two disease sites within 5 d before the first and second avelumab treatments. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the disease control rate (DCR), defined as a confirmed complete or partial response of any duration, or stable disease/non-complete response/non-progressive disease for ≥6 mo (Prostate Cancer Clinical Trials Working Group 3-modified Response Evaluation Criteria in Solid Tumours version 1.1). Secondary endpoints were the objective response rate (ORR), radiographic progression-free survival (rPFS), overall survival (OS), and safety. DCR and ORR were calculated using the Clopper-Pearson exact binomial method. RESULTS AND LIMITATIONS: Thirty-one evaluable men were enrolled (median age 71 yr, 71% with ≥2 prior mCRPC therapy lines, 81% with >5 total metastases). The DCR was 48% (15/31; 95% confidence interval [CI] 30-67%) and ORR was 31% (five of 16; 95% CI 11-59%). The ORR in nonirradiated lesions was 33% (four of 12; 95% CI 10-65%). Median rPFS was 8.4 mo (95% CI 4.5-not reached [NR]) and median OS was 14.1 mo (95% CI 8.9-NR). Grade 3-4 treatment-related adverse events occurred in six patients (16%), with three (10%) requiring high-dose corticosteroid therapy. Plasma androgen receptor alterations were associated with lower DCR (22% vs 71%, p = 0.13; Fisher's exact test). Limitations include the small sample size and the absence of a control arm. CONCLUSIONS: Avelumab with SABR demonstrated encouraging activity and acceptable toxicity in treatment-refractory mCRPC. This combination warrants further investigation. PATIENT SUMMARY: In this study of men with advanced and heavily pretreated prostate cancer, combining stereotactic radiotherapy with avelumab immunotherapy was safe and resulted in nearly half of patients experiencing cancer control for 6 months or longer. Stereotactic radiotherapy may potentially improve the effectiveness of immunotherapy in prostate cancer.

Data-independent acquisition mass spectrometry for site-specific glycoproteomics characterization of SARS-CoV-2 spike protein.

The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of the SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low-abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low-abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA outperforms DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development.

Glycosylation of Serum Clusterin in Wild-Type Transthyretin-Associated (ATTRwt) Amyloidosis: A Study of Disease-Associated Compositional Features Using Mass Spectrometry Analyses.

Wild-type transthyretin-associated (ATTRwt) amyloidosis is an age-related disease that causes heart failure in older adults. This disease frequently features cardiac amyloid fibril deposits that originate from dissociation of the tetrameric protein, transthyretin (TTR). Unlike hereditary TTR (ATTRm) amyloidosis, where amino acid replacements destabilize the native protein, in ATTRwt amyloidosis, amyloid-forming TTR lacks protein sequence alterations. The initiating cause of fibril formation in ATTRwt amyloidosis is unclear, and thus, it seems plausible that other factors are involved in TTR misfolding and unregulated accumulation of wild-type TTR fibrils. We believe that clusterin (CLU, UniProtKB P10909), a plasma circulating glycoprotein, plays a role in the pathobiology of ATTRwt amyloidosis. Previously, we have suggested a role for CLU in ATTRwt amyloidosis based on our studies showing that (1) CLU codeposits with non-native TTR in amyloid fibrils from ATTRwt cardiac tissue, (2) CLU interacts only with non-native (monomeric and aggregated) forms of TTR, and (3) CLU serum levels in patients with ATTRwt are significantly lower compared to healthy controls. In the present study, we provide comprehensive detail of compositional findings from mass spectrometry analyses of amino acid and glycan content of CLU purified from ATTRwt and control sera. The characterization of oligosaccharide content in serum CLU derived from patients with ATTRwt amyloidosis is novel data. Moreover, results comparing CLU oligosaccharide variations between patient and healthy controls are original and provide further evidence for the role of CLU in ATTRwt pathobiology, possibly linked to disease-specific structural features that limit the chaperoning capacity of CLU.

Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty.

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

Multi-task learning sparse group lasso: a method for quantifying antigenicity of influenza A(H1N1) virus using mutations and variations in glycosylation of Hemagglutinin.

BACKGROUND: In addition to causing the pandemic influenza outbreaks of 1918 and 2009, subtype H1N1 influenza A viruses (IAVs) have caused seasonal epidemics since 1977. Antigenic property of influenza viruses are determined by both protein sequence and N-linked glycosylation of influenza glycoproteins, especially hemagglutinin (HA). The currently available computational methods are only considered features in protein sequence but not N-linked glycosylation. RESULTS: A multi-task learning sparse group least absolute shrinkage and selection operator (LASSO) (MTL-SGL) regression method was developed and applied to derive two types of predominant features including protein sequence and N-linked glycosylation in hemagglutinin (HA) affecting variations in serologic data for human and swine H1N1 IAVs. Results suggested that mutations and changes in N-linked glycosylation sites are associated with the rise of antigenic variants of H1N1 IAVs. Furthermore, the implicated mutations are predominantly located at five reported antibody-binding sites, and within or close to the HA receptor binding site. All of the three N-linked glycosylation sites (i.e. sequons NCSV at HA 54, NHTV at HA 125, and NLSK at HA 160) identified by MTL-SGL to determine antigenic changes were experimentally validated in the H1N1 antigenic variants using mass spectrometry analyses. Compared with conventional sparse learning methods, MTL-SGL achieved a lower prediction error and higher accuracy, indicating that grouped features and MTL in the MTL-SGL method are not only able to handle serologic data generated from multiple reagents, supplies, and protocols, but also perform better in genetic sequence-based antigenic quantification. CONCLUSIONS: In summary, the results of this study suggest that mutations and variations in N-glycosylation in HA caused antigenic variations in H1N1 IAVs and that the sequence-based antigenicity predictive model will be useful in understanding antigenic evolution of IAVs.

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus.

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PV-infected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

Why Glycosylation Matters in Building a Better Flu Vaccine.

Low vaccine efficacy against seasonal influenza A virus (IAV) stems from the ability of the virus to evade existing immunity while maintaining fitness. Although most potent neutralizing antibodies bind antigenic sites on the globular head domain of the IAV envelope glycoprotein hemagglutinin (HA), the error-prone IAV polymerase enables rapid evolution of key antigenic sites, resulting in immune escape. Significantly, the appearance of new N-glycosylation consensus sequences (sequons, NXT/NXS, rarely NXC) on the HA globular domain occurs among the more prevalent mutations as an IAV strain undergoes antigenic drift. The appearance of new glycosylation shields underlying amino acid residues from antibody contact, tunes receptor specificity, and balances receptor avidity with virion escape, all of which help maintain viral propagation through seasonal mutations. The World Health Organization selects seasonal vaccine strains based on information from surveillance, laboratory, and clinical observations. Although the genetic sequences are known, mature glycosylated structures of circulating strains are not defined. In this review, we summarize mass spectrometric methods for quantifying site-specific glycosylation in IAV strains and compare the evolution of IAV glycosylation to that of human immunodeficiency virus. We argue that the determination of site-specific glycosylation of IAV glycoproteins would enable development of vaccines that take advantage of glycosylation-dependent mechanisms whereby virus glycoproteins are processed by antigen presenting cells.

IQcat: multiplexed protein quantification by isoelectric QconCAT.

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (∼2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.

Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin.

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.

ChromEval: a software application for the rapid evaluation of HPLC system performance in proteomic applications.

Mass spectrometry-based proteomics is typically performed using high performance liquid chromatography (HPLC) to introduce peptides into the instrument via electrospray ionization. A variety of configurations exist with varying degrees of precision and cost, but the ultimate goal is the reproducible delivery of peptides in well-separated elution peaks. It is well-known that the quality of chromatography can have a dramatic effect on sample identification as well as run-to-run reproducibility, which is especially important for quantitative analyses. Despite the importance of the HPLC system for both shotgun and targeted proteomics, there are few tools available to monitor HPLC performance. In this paper, we describe a new open-source software application, named ChromEval, to allow rapid assessment of HPLC performance, as well as to provide other metrics of mass spectrometer performance, including mass accuracy calibration. ChromEval permits the user to visually monitor the elution of a set of standard peptides in quality control runs interspersed among a regular workflow. To perform these tasks, ChromEval searches mzXML files using Tandem and presents the peptide results in a graphical user interface (GUI) that allows fast assessment of chromatography by visualization of superimposed elution peaks. This tool facilitates the identification and troubleshooting of chromatography problems such as retention time shifts and variance in sample loading due to autosampler error. It also provides crude but consistent metrics of instrument performance including mass accuracy calibration and number of peptides identified from the standard mixture. ChromEval generates easily interpretable data quickly and thereby enables go/no-go decision making during intensive instrument operation.