Quantitative dynamics comparison between SureQuant pTyr, PRM, and DDA.

Targeted tyrosine phosphorylated peptides, method parameters, and file map of raw data files.

Quantitative reproducibility comparison between SureQuant pTyr and DDA.

Clinical data for tumor specimens analyzed.

Custom pathway and substrate-kinase libraries for tumor-specific pathway enrichment analyses.

Abstract The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS enhanced quant of single cell spectra) that makes use of fast realtime searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at an MS2 injection time of 750 ms using a 2 h gradient.

Abstract Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide–HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

LC-MS-based proteomics analysis has shown to be a powerful analytical tool for identification and quantification of thousands of proteins in complex biological samples. Moving forward from discovery to targeted quantitation in proteomics, there is a need for a robust mass spectrometry system and methods that provide reproducibility needed to analyze thousands of samples without compromising on coverage and quantitation performance with ease of use for any level of analytical expertise. Here we present the Thermo ScientificTM Orbitrap ExplorisTM 480 mass spectrometer coupled to the Thermo ScientificTM FAIMS ProTM Interface, a high-field asymmetric waveform ion mobility spectrometry (FAIMS) system for proteomics applications. The performance of this small benchtop mass spectrometer is evaluated in data-dependent acquisition (DDA) mode for sample injection amounts of just a single HeLa cell up to 5000 HeLa cells (~1 ug), providing the sensitivity and performance required for analysis of large numbers of samples without compromising on sensitivity and proteome coverage. To demonstrate the sensitivity of the instrument, we analyzed proteins from a single HeLa cell, as well as bulk digest equal to single-cell protein levels. This instrument sensitivity enables identification of ~7000 protein groups from only a 200 ng of bulk HeLa digest and ~800 protein groups from a single HeLa cell in a 2 hour LC gradient LC-MS analysis. The method performance at 1 ug sample injection was also evaluated across different instruments located in different laboratories. TECHNICAL NOTE 65944

Abstract Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry–based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed “SureQuant pTyr,” coupling low input pTyr enrichment with a panel of isotopically labeled internal standard peptides to guide data acquisition of low-abundance tyrosine phosphopeptides. SureQuant pTyr allowed for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, improving the robustness and usability of targeted mass spectrometry assays. We established the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient-specific oncogenic-driving mechanisms. While in some cases pTyr profiles aligned with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlighted instances of new insights gained using pTyr characterization and emphasized the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enables pTyr-based measurements for applications in precision medicine. Significance: SureQuant pTyr is a mass spectrometry–based targeted method that enables sensitive and selective targeted quantitation of several hundred low-abundance tyrosine phosphorylated peptides commonly dysregulated in cancer, including oncogenic signaling networks.

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by highintensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC-MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.

In the last decade, single-cell RNA sequencing has advanced our understanding of transcript heterogeneity. Currently, there are strong efforts to enable single-cell proteomic analysis using mass spectrometry (MS)-based workflows. While the analysis of single-cell-sized aliquots from bulk-prepared tryptic digests has been demonstrated, only recently have label-free strategies been reported for profiling hundreds of proteins from single mammalian cells. Further development in sample processing, separations, MS and data analysis seems necessary to realize single-cell proteomics with greater depth of coverage and quantitative accuracy. Here we introduce an improved LC separation coupled to the new Thermo ScientificTM Orbitrap EclipseTM TribridTM mass spectrometers with a Thermo ScientificTM FAIMS ProTM interface to increase proteome coverage for single mammalian cells.

The rapidly increasing adoption of high-resolution accurate-mass methods in analytical laboratories has fueled demand for instruments that combine high performance and reliability with small size and greater ease-of-use. This paper presents the major design principles that are driving the evolution of the hybrid quadrupole-Orbitrap instrument architecture to enable a greater range of applications and users. These principles may be summarized as follows: better usage of physical space and better access for service by means of size reduction of pumping and ion optics; expanded use of technologies from electronics in ion-optical design; flexibility in performance via modularity of design of the hardware and software components; and, harmonization of interfaces with other instruments to facilitate sharing and transferability of analytical workflows. The design of a novel family of hybrid mass spectrometers is described in detail, and performance evaluation is carried out on a wide variety of samples for its three representatives: the Orbitrap Exploris 120, Orbitrap Exploris 240 and Orbitrap Exploris 480 mass spectrometers.The new instrument family is shown to offer compelling potential not only for high-end proteomics and biopharmaceutical applications, but also for screening, trace, targeted and clinical analysis by liquid chromatography/mass spectrometry methods.

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

Epilepsy affects millions of individuals worldwide and many cases are pharmacoresistant. Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region. Phenotypes include a high rate of pharmacoresistant epilepsy. We developed a Dup15q model in Drosophila melanogaster that recapitulates seizures in Dup15q by over-expressing fly Dube3a or human UBE3A in glial cells, but not neurons, implicating glia in the Dup15q epilepsy phenotype. We compared Dube3a overexpression in glia (repo>Dube3a) versus neurons (elav>Dube3a) using transcriptomics and proteomics of whole fly head extracts. We identified 851 transcripts differentially regulated in repo>Dube3a, including an upregulation of glutathione S-transferase (GST) genes that occurred cell autonomously within glial cells. We reliably measured approximately 2,500 proteins by proteomics, most of which were also quantified at the transcript level. Combined transcriptomic and proteomic analysis revealed an enrichment of 21 synaptic transmission genes downregulated at the transcript and protein in repo>Dube3a indicating synaptic proteins change in a cell non-autonomous manner in repo>Dube3a flies. We identified 6 additional glia originating bang-sensitive seizure lines and found upregulation of GSTs in 4 out of these 6 lines. These data suggest GST upregulation is common among gliopathic seizures and may ultimately provide insight for treating epilepsy.

Isobaric tagging strategies using Thermo ScientificTM Tandem Mass TagsTM (TMTTM) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo ScientificTM Orbitrap EclipseTM TribridTM mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.