Serial and multi-level proteome analysis for microscale protein samples

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The "one-pot" affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

High resolution data-independent acquisition with electron transfer dissociation mass spectrometry: Multiplexed analysis of post-translationally modified proteins

Liquid chromatography(LC) tandem mass spectrometry (MS/MS) isone of the widely used proteomic techniques to study the alterationsoccurring at the protein level as well as post-translational modifications(PTMs) of proteins that are relevant to different physiological orpathological statuses. The mass spectrometric analysis of peptidesdigested from proteins (bottom-up proteomics) has emerged as one ofthe major approaches for proteomics. In this approach, proteins arefirst cleaved by one or more proteases into peptides for MS analysis,and peptides with PTMs are further enriched, followed by the LC-MS/MSanalysis. To achieve a reproducible and quantitative proteomic characterization,a well-established protease digestion and PTM peptide enrichment protocolis critical. In this study, we developed AUTO-SP, a sample preparationplatform providing automated protocols for BCA analysis, protein digestion,and PTM enrichment for protein and PTM analyses. We utilized patient-derivedxenograft (PDX) breast cancer tumor tissues (basal-like and luminalsubtypes) to demonstrate the efficacy of AUTO-SP. The protein amountwas quantified, and proteins were further digested by using AUTO-SPfor each PDX sample. Based on the data-independent acquisition (DIA)-MSdata, we observed that samples of the same breast cancer subtypeswere highly correlated (≥0.98). Additionally, >25,000 phosphopeptidesand >14,000 ubiquitinated peptides were identified in the PDX sampleswhen using AUTO-SP for PTM enrichment, while unique pathways wereenriched from the differentially expressed ubiquitinated peptidesof basal-like and luminal subtypes. AUTO-SP demonstrated its efficacyto provide a reliable and reproducible sample preparation procedurefor MS-based proteomic and PTM analyses.

Macrophages secrete diverse signaling proteins critical for intercellular communication and immune responses, processes tightly regulated by post-translational modifications (PTMs). Itaconate, an immunoregulatory metabolite produced in macrophages, induces widespread intracellular protein modification (itaconation), affecting pathways like the KEAP1-NRF2 axis and glycolysis. However, the impact of itaconation on the extracellular proteome and signaling remains poorly characterized. Herein, we introduce PTM-based secretome profiling (PBSP), a novel approach to identify secreted proteins bearing specific PTMs. The method employs a bioorthogonal probe to label modified proteins in live cells, followed by enrichment of labeled proteins from the culture medium upon secretion. We established a streamlined chemoproteomic workflow integrating spintip-based affinity purification (FISAP) with data-independent acquisition (DIA) mass spectrometry for enhanced sensitivity and coverage. This identified 818 macrophage-secreted itaconated proteins, among which 447 are exosome-dependent. Further biochemical analysis revealed that itaconation of Cys239 on FYN (a tyrosine kinase) enhances its kinase activity in macrophages. We finally demonstrate PBSP’s versatility by profiling secreted proteins with other PTMs, including fumarate-induced succination. PBSP provides a powerful platform to explore PTM roles in protein secretion, offering insights into PTMs’ regulatory functions in cell-cell communication.

Rapid high-throughput antibody analysis using microwave-assisted digestion.

Advances in library-based methods for peptide detection from data-independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high-quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico, but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate-controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for accurate peptide detection and quantitation in wide-window DIA data. We compare the results of this search workflow to other library-free approaches and demonstrate that our search is competitive in terms of accuracy and sensitivity. These results demonstrate that the proposed workflow has the capacity to generate spectral libraries while avoiding the limitations of other methods.

Post-translational modification (PTM) of protein lysine residues by NƐ-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Epiproteomic Landscape and Histone Code of Cutaneous T-Cell Lymphoma/Sézary Syndrome

Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis

This chapter introduces computational methods used in mass spectrometry-based proteomics, including those for addressing the critical problems such as peptide identification and protein inference, peptide and protein quantification, characterization of posttranslational modifications (PTMs), and data-independent acquisitions (DIA). The chapter concludes with emerging applications of proteomic techniques, such as metaproteomics, glycoproteomics, and proteogenomics.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.

Protein degradation has become an area of focus for modern research due to the technological advancement of targeted protein degradation as a highly specific and selective therapeutic approach [1]. However, as can be said of most scientific discoveries, the cell itself has long utilized and fine-tuned this regulatory process to create intricate layers of regulation surrounding protein degradation to attune the proteome to intracellular and extracellular cues. Thus far, the answer to this question has indicated that post-translational modifications (PTMs) on target proteins are responsible for tuning protein degradation to the cell's needs [2]. As our understanding of protein degradation delves further into the mechanistic and biological implications of the relationship between the cellular environment, PTMs, and protein degradation, further questions are uncovered regarding why PTMs have evolved to play these central roles in protein degradation. This hypothesis by Hwang et al., [3] takes a refreshing perspective on this next step: Can the ubiquitination machinery itself be directly subject to environmental cues to add an additional regulatory layer to protein degradation? Protein degradation largely occurs through the ubiquitin-proteasome system (UPS). The UPS utilizes a three-step enzymatic cascade to processively attach ubiquitin (Ub) onto surface lysine residues of proteins targeted for degradation. The ubiquitination process is highly coordinated, thereby allowing for regulation at almost every step before, during, and after the ubiquitination cascade. The most common regulatory step occurs at ubiquitin attachment, wherein E3 ligase complexes function to coordinate transfer of the ubiquitin from an E2 ubiquitin-conjugating enzyme onto the target protein. These complexes are often made up of multiple adaptor and scaffold proteins, again indicating the level of regulation required for ubiquitination. Cullin-RING ligases (CRLs), the majority of known E3 ligases, consist of a Cullin scaffold protein and a RING adaptor protein which functions to bind the E2-Ub complex. Families of CRLs are characterized based on the other proteins that make up the complex. Skp1-cullin1-Fbox (SCF) are a subset of CRLs that utilize adaptor protein Skp1 to bind an F-box protein (FBP) bound to the target protein [4]. Intuitively, the recruitment of the target protein to the SCF complex via FBPs is crucial for protein ubiquitination via SCF complexes. This article posits the hypothesis that FBPs function as environmental sensors to control stress response mechanisms, particularly in response to heavy metals [3]. FBPs are well-evidenced to respond to various PTMs (including but not limited to phosphorylation, methylation, and acetylation), which are arguably all ways in which the cell can transmit environmental signals to protein degradation. The authors emphasis of hydroxylation [5] within this context is, therefore, an expansion on well-known regulatory processes dictating how PTMs allow FBP-target protein interactions to remain sensitive to environmental perturbations. The author's expansion into heavy metal sensing, however, is an exciting new hypothesis that is so novel that it is potentially under-evidenced. Canonically, the FBP is made up of two domains: (1) an F-box domain highly conserved for the interaction to adaptor protein Skp1 and (2) a protein interaction domain that binds the protein targeted for degradation. The authors hypothesize that the F-box domain may also sense heavy metals with functional implications to protein degradation, in addition to being an anchor to Skp1 and the rest of the SCF complex. The foundation for this hypothesis is found in the examples of yeast FBPs outlined within the article [6, 7], leading to the suggestion that human FBPs may also contain these functions. However, initial phenotypic verification of this hypothesis is not currently available. Correlative studies demonstrating statistically significant links between SCF complex activity and/or stability in the presence of various metals via cell-wide techniques (i.e., proteomics) could address this issue. In summary, the authors present an intriguing next step for the field of protein homeostasis: the integration of findings from other species to delve into domains and mechanisms long believed to contain just one simple function. This hypothesis builds on a strong foundation of FBPs as conditionally sensitive via PTM-mediated or -hindered protein-protein interactions with proteins targeted for degradation and looks ahead to new possibilities for the regulation of protein degradation. This article comments on the hypothesis paper by Jiwon Hwang, Linda Lauinger, and Peter Kaiser https://doi.org/10.1002/bies.202400249. The authors declare no conflicts of interest.