Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 µg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 µg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

A Tip-Based Workflow for Sensitive IMAC-Based Low Nanogram Level Phosphoproteomics.

Analyzing the phosphoproteome at nanoscale poses a significant challenge, mainly due to the substantial sample loss from nonspecific surface adsorption during the enrichment of low stoichiometric phosphopeptides. Here, we describe a tandem tip-based phosphoproteomics sample preparation method capable of sequential sample cleanup and enrichment without the need for additional sample transfer, thereby minimizing sample loss. Integration of this method to our recently developed SOP (surfactant-assisted one-pot sample preparation) and iBASIL (improved boosting to amplify signal with isobaric labeling) approaches creates a streamlined workflow, enabling sensitive, high-throughput nanoscale phosphoproteomics measurements.

High-throughput relative quantification of fatty acids by 12-plex isobaric labeling and microchip capillary electrophoresis - Mass spectrometry.

BACKGROUND: Fatty acids (FAs) are essential cellular components and play important roles in various biological processes. Importantly, FAs produced by microorganisms from renewable sugars are considered sustainable substrates for biodiesels and oleochemicals. Their complex structures and diverse functional roles in biochemical processes necessitate the development of efficient and accurate methods for their quantitative analysis. RESULTS: Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric N,N-dimethyl leucine-derivatized ethylenediamine (DiLeuEN) labeling and microchip capillary electrophoresis-mass spectrometry (CE-MS). This method enables simultaneous quantification of 12 samples in a single MS analysis. DiLeuEN labeling introduced tertiary amine center structure into FAs, which makes them compatible with the positive mode separation of commercial microchip CE systems and further improves the sensitivity. The CE separation parameters were optimized, and the quantification accuracy was assessed using FA standards. Microchip CE-MS detection exhibited high sensitivity with a femtomole level detection limit and a total analysis time within 8 min. Finally, the applicability of our method to complex biological samples was demonstrated by analyzing FAs produced by four industrially relevant yeast strains (Saccharomyces cerevisiae, Yarrowia lipolytica YB-432, Yarrowia lipolytica Po1f and Rhodotorula glutinis). The analysis time for each sample is less than 1 min. SIGNIFICANCE: This work addresses the current challenges in the field by introducing a method that combines microchip-based capillary electrophoresis separation with multiplex isobaric labeling. Our method not only offers remarkable sensitivity and rapid analysis speed but also the capability to quantify fatty acids across multiple samples simultaneously, which holds significant potential for extensive application in FA quantitative studies in diverse research areas, promising an enhanced understanding of FA functions and mechanisms.

12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.

Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC - MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.

An efficient, amine-specific iTRAQ labeling method improves the peptide and protein identification rates.

Isotope tags for relative and absolute quantification (iTRAQ) are among the most widely used proteomics quantification techniques. These tags can be rapidly coupled to the primary amines of proteins/peptides through chemical reactions under mild conditions, making this technique universally applicable to any kind of sample. However, iTRAQ reagents also partially react with the hydroxyl groups of serine, threonine and tyrosine residues, particularly when these residues coexist with a histidine residue in the same peptide. This overlabeling of peptides causes systematic biases and significantly compromises protein/peptide identification rates. In this study, we report a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high amine labeling efficiency. The impacts of reaction temperature, reactant concentrations, reaction time, buffer compositions, and pH on iTRAQ labeling performance were investigated in-depth. In a comparison experiment between our method and the standard labeling method provided by the iTRAQ manufacturer, our method reduced the number of overlabeled peptides by 55-fold while achieving comparable amine labeling efficiency. This improvement allowed our method to eliminates the systematic bias against histidyl- and hydroxyl-containing peptides, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins. SIGNIFICANCE: In addition to amines, the hydroxyl groups in serine, threonine, and tyrosine residues can also partially labeled by iTRAQ reagents, which leads to systematic biases and significantly compromises the analytical sensitivity. To address this issue, we developed a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high labeling efficiency of amines. When benchmarking our method against the standard method provided by the reagent manufacturer, our method achieved comparable labeling efficiency but reduced the overlabeled species by 55-fold. This significant improvement eliminated the systematic biases, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins, demonstrating its superior performance and potential to enhance proteome quantification using iTRAQ labeling.

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.

GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

Quantification Quality Control Emerges as a Crucial Factor to Enhance Single-Cell Proteomics Data Analysis.

Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similar to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies.

An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage, Quantitative Accuracy and Precision.

Tandem mass tags (TMT) are widely used in proteomics to simultaneously quantify multiple samples in a single experiment. The tags can be easily added to the primary amines of peptides/proteins through chemical reactions. In addition to amines, TMT reagents also partially react with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the analytical sensitivity and precision. Under alkaline conditions, reducing the TMT molar excess can partially mitigate overlabeling of histidine-free peptides, but has a limited effect on peptides containing histidine and hydroxyl groups. Here, we present a method under acidic conditions to suppress overlabeling while efficiently labeling amines, using only one-fifth of the TMT amount recommended by the manufacturer. In a deep-scale analysis of a yeast/human two-proteome sample, we systematically evaluated our method against the manufacturer's method and a previously reported TMT-reduced method. Our method reduced overlabeled peptides by 9-fold and 6-fold, respectively, resulting in the substantial enhancement in peptide/protein identification rates. More importantly, the quantitative accuracy and precision were improved as overlabeling was reduced, endowing our method with greater statistical power to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively. Mass spectrometric data have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD047052.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE-PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

Recent progress in quantitative phosphoproteomics.

INTRODUCTION: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events. AREAS COVERED: State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples. EXPERT OPINION: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

Quantitative Proteomics Using Isobaric Labeling: A Practical Guide / 基因组蛋白质组与生物信息学报·英文版

In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.

Screening of Differential Expression Proteins in Rat Brain Tissues after DAI by iTRAQ-LC-MS/MS / 法医学杂志

Objective To screen for the differential expression proteins in brain tissues of SD rat after diffuse axonal injury (DAI) by isobaric tag for relative and absolute quantification-liquid chromatographmass spectrometer/mass spectrometer (iTRAQ-LC-MS/MS),and to explore potential biomarkers available for the diagnosis of DAI.Methods Animal models of DAI were established with the Marmarou method as reference,and the subjects were divided into blank control group (n=4),sham strike group (n=4) and fatal strike group (n=4),respectively.The proteins in rat brain tissues were detected by iTRAQ-LC-MS/MS,and bioinformatics analysis and verification were performed on the results and screened for the differential expression proteins.Results A total of 2 016 proteins were identified and quantified.The bioinformatics analysis revealed that the proteins had wide distribution and function,and participated in different biological processes.There were 16 proteins showed differential expression in fatal strike group,including one up-regulated expression protein and 15 down-regulated expression proteins.The results of iTRAQLC-MS/MS were confirmed by Western blotting method.Conclusion Multiple differential expression proteins in rat brain tissues after DAI can be screened by iTRAQ-LC-MS/MS.This not only indicates a research direction for exploring the pathogenesis of DAI,but also provides potential biomarkers available for the diagnosis of DAI.

Novel Algorithm for Identification and Quantification of Proteins Based on Strategy of Isobaric Peptide Termini Labelling / 分析化学

Isobaric peptide termini labeling ( IPTL) is a technology which uses light and heavy isotopes to label C-terminus and N-terminus of peptides. As the masses of labeled peptides are equivalent, the complexity of sample is low when analyzing MS data produced by this technology. Besides, paired b and y ions are helpful while analyzing MS/MS data in this kind of experiments. On the basis of this, a novel scoring algorithm, all ions scoring algorithm (AISA), has been designed for IPTL experiments. The information of quantification and qualification can be acquired at the same time using AISA. On Q-Exactive HeLa 2D RPLC dataset, peptide spectrum matches ( PSMs) , distinct peptides and protein groups identified by AISA are 15%, 26%and 22% higher than Morpheus. On human-HCC-HL dataset, PSMs, distinct peptides and protein groups identified by AISA are 24%, 39% and 27% higher than Morpheus. Quantification ratio on Q-Exactive HeLa and human-HCC-HL datasets are 1. 18 and 0. 90, respectively, which are very close to 1. Besides, quantification ratios between 0. 5 and 2. 0 are 91% and 94%, respectively.

Novel Algorithm for Identification and Quantification of Proteins Based on Strategy of Isobaric Peptide Termini Labelling / 分析化学

Isobaric peptide termini labeling ( IPTL) is a technology which uses light and heavy isotopes to label C-terminus and N-terminus of peptides. As the masses of labeled peptides are equivalent, the complexity of sample is low when analyzing MS data produced by this technology. Besides, paired b and y ions are helpful while analyzing MS/MS data in this kind of experiments. On the basis of this, a novel scoring algorithm, all ions scoring algorithm (AISA), has been designed for IPTL experiments. The information of quantification and qualification can be acquired at the same time using AISA. On Q-Exactive HeLa 2D RPLC dataset, peptide spectrum matches ( PSMs) , distinct peptides and protein groups identified by AISA are 15%, 26%and 22% higher than Morpheus. On human-HCC-HL dataset, PSMs, distinct peptides and protein groups identified by AISA are 24%, 39% and 27% higher than Morpheus. Quantification ratio on Q-Exactive HeLa and human-HCC-HL datasets are 1. 18 and 0. 90, respectively, which are very close to 1. Besides, quantification ratios between 0. 5 and 2. 0 are 91% and 94%, respectively.