Wei Jun Qian

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

Selected reaction monitoring (SRM) – also known as multiple reaction monitoring (MRM) – has emerged as a promising high‐throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for, e.g. detecting low‐abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low‐abundance signaling proteins in cells or tissues. Herein, we review recent advances in methods and technologies, including front‐end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications, as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low‐abundance proteins at low‐ to sub‐ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.

Liver-Derived Systemic Factors Drive β Cell Hyperplasia in Insulin-Resistant States

Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates β cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary inxa0vivo parabiosis and transplantation assays, as well as inxa0vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces β cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β cell proliferation in exxa0vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β cell growth factor(s) in insulin-resistant states.

SerpinB1 Promotes Pancreatic β Cell Proliferation

Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model that manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β cell proliferation in humans, mice, and zebrafish. Small-molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β cells, and mice lacking serpinB1 exhibit attenuated β cell compensation in response to insulin resistance. Finally, SerpinB1 treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β cell mass in patients with diabetes.

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomics biomarker discovery

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.

Quantitative proteomic approaches for studying phosphotyrosine signaling

Protein tyrosine phosphorylation is a fundamental mechanism for controlling many aspects of cellular processes, as well as aspects of human health and diseases. Compared with phosphoserine and phosphothreonine, phosphotyrosine signaling is more tightly regulated, but often more challenging to characterize, due to significantly lower levels of tyrosine phosphorylation (i.e., a relative abundance of 1800:200:1 was estimated for phosphoserine/phosphothreonine/phosphotyrosine in vertebrate cells). In this review, we outline recent advances in analytical methodologies for enrichment, identification and accurate quantitation of tyrosine-phosphorylated proteins and peptides. Advances in antibody-based technologies, capillary liquid chromatography coupled with mass spectrometry, and various stable isotope labeling strategies are discussed, as well as non-mass spectrometry-based methods, such as those using protein/peptide arrays. As a result of these advances, powerful tools now have the power to crack signal transduction codes at the system level, and provide a basis for discovering novel drug targets for human diseases.

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry.

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.

Advances in targeted proteomics and applications to biomedical research

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data‐independent acquisition (DIA) with targeted data extraction on fast scanning high‐resolution accurate‐mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale quantification of hundreds of target proteins are discussed.

Quantitative Proteome Mapping of Nitrotyrosines

An essential first step in the understanding disease and environmental perturbations is the early and quantitative detection of the increased levels of the inflammatory marker nitrotyrosine, as compared with its endogenous levels within the tissue or cellular proteome. Thus, methods that successfully address a proteome-wide quantitation of nitrotyrosine and related oxidative modifications can provide early biomarkers of risk and progression of disease, as well as effective strategies for therapy. Multidimensional separations LC coupled with tandem mass spectrometry (LC-MS/MS) has, in recent years, significantly expanded our knowledge of human (and mammalian model system) proteomes, including some nascent work in identification of posttranslational modifications. This chapter discusses the application of LC-MS/MS for quantitation and identification of nitrotyrosine-modified proteins within the context of complex protein mixtures presented in mammalian proteomes.

Endogenous 3,4-Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine LINKS TO MITOCHONDRIALLY DERIVED OXIDATIVE STRESS VIA HYDROXYL RADICAL

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.

Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps.