TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples.

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing.

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

TMTpro: Design, Synthesis, and Initial Evaluation of a Proline-Based Isobaric 16-Plex Tandem Mass Tag Reagent Set.

The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis.

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

An integrated workflow for multiplex CSF proteomics and peptidomics-identification of candidate cerebrospinal fluid biomarkers of Alzheimer's disease.

Many disease processes in the brain are reflected in the protein composition of the cerebrospinal fluid (CSF). In addition to proteins, CSF also contains a large number of endogenous peptides whose potential as disease biomarkers largely remains to be explored. We have developed a novel workflow in which multiplex isobaric labeling is used for simultaneous quantification of endogenous CSF peptides and proteins by liquid chromatography coupled with mass spectrometry. After the labeling of CSF samples, endogenous peptides are separated from proteins by ultrafiltration. The proteins retained on the filters are trypsinized, and the tryptic peptides are collected separately. We evaluated this technique in a comparative pilot study of CSF peptide and protein profiles in eight patients with Alzheimer's disease (AD) and eight nondemented controls. We identified several differences between the AD and control group among endogenous peptides derived from proteins known to be associated with AD, including neurosecretory protein VGF (ratios AD/controls 0.45-0.81), integral membrane protein 2B (ratios AD/controls 0.72-0.84), and metallothionein-3 (ratios AD/controls 0.51-0.61). Analysis of tryptic peptides identified several proteins that were altered in the AD group, some of which have previously been reported as changed in AD, for example, VGF (ratio AD/controls 0.70).

Pharmacoproteomic investigation into antidepressant response in two mouse inbred strains.

In this study, we present a pharmacoproteomic investigation of response to antidepressants two inbred strains. Our aim was to uncover molecular mechanisms underlying antidepressant action and identify new biomarkers to determine therapeutic response to two antidepressants with proven efficacy in the treatment of depression but divergent mechanisms of action. Mice were treated with the pro-noradrenergic drug nortriptyline, the pro-serotonergic drug escitalopram or saline. Quantitative proteomic analyses were undertaken on hippocampal tissue from a study design that used two inbred mouse strains, two depressogenic protocols and a control condition, (maternal separation, chronic mild stress, control), two antidepressant drugs and two dosing protocols. The proteomic analysis was aimed at the identification of specific drug-response markers. Complementary approaches, 2DE and isobaric tandem mass tagging (TMT), were applied to the selected experimental groups. To investigate the relationship between proteomic profiles, depressogenic protocols and drug response, 2DE and TMT data sets were analysed using multivariate methods. The results highlighted significant strain- and stress-related differences across both 2DE and TMT data sets and identified the three gene products involved in serotonergic (PXBD5, YHWAB, SLC25A4) and one in noradrenergic antidepressant action (PXBD6).

Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked glycans.

N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 µM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.

Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.

Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a (15)N for a (13)C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a (13)C was exchanged for a (15)N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the (13)C- and (15)N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex.

Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach.

Isobaric labeling reagents such as Tandem Mass Tags (TMT(R)) enable the genome-wide quantification of protein expression levels under different conditions using a gel-free MS/MS-based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron. Expression of 35 proteins was found to be induced or repressed under iron-limiting conditions, including 11 proteins whose ORFs were not previously identified in DNA array studies as being regulated by iron availability at the transcriptional level. These 11 proteins include proteins likely involved in iron metabolism.

Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags.

A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.

In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

TMT labelling for the quantitative analysis of adaptive responses in the meningococcal proteome.

In addition to standard gel-based proteomic approaches, gel-free approaches using isobaric label reagents, such as Tandem Mass Tags (TMT), provide a straightforward method for studying adaptations in microbial proteomes to changing environmental conditions. This approach does not have the known difficulties of 2-D gel electrophoresis with proteins of extreme biochemical properties. The workflow described here was designed to study adaptive responses in bacteria and has been applied to study the response of meningococci to iron limitation. The supplemental use of western blotting allows the confirmation of certain changes in protein abundance identified within the TMT study.

Breast Cancer Proteomics - Differences in Protein Expression between Estrogen Receptor-Positive and -Negative Tumors Identified by Tandem Mass Tag Technology.

BACKGROUND: Proteomic analysis has become an effective tool in breast cancer research. In this study, we applied the new gel-free tandem mass tag (TMT) reference method for the first time in breast cancer. MATERIALS AND METHODS: Proteomic analysis was used to compare 10 estrogen receptor (ER)-positive and 10 ER-negative samples. The results of the proteomic approach were validated by Western blot, immunohistochemistry and gene expression analysis. RESULTS: 17 proteins with significant differences in expression were identified. 13 proteins were overexpressed in ER-negative tumors and 4 were overexpressed in ER-positive samples. All these proteins were characterized by relatively high cellular abundance. CONCLUSIONS: Our results demonstrate that the gel-free TMT approach allows the quantification of differences in protein expression levels. Further improvement of the sensitivity by subfractionation of the tissue should allow also the identification of low-abundance proteins and might lead to the use of this method in breast cancer research.

Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring.

Tandem Mass Tags (TMT) are suited to both global and targeted quantitation approaches of proteins and peptides. Different versions of these tags allow for the generation of both isobaric and isotopic sets of reagents sharing the same common structure. This feature allows for a straightforward transfer of data obtained during discovery studies into targeted investigations. In prior discovery studies, an isobaric set of these reagents was used to identify Neisseria meningitidis proteins expressed under iron-limitation. Here, we apply isotopic versions of those reagents in combination with single reaction monitoring to verify selected candidates found to be differentially regulated in these discovery studies, representing both well-known and novel iron-regulated proteins, such as the MtrCDE drug efflux pump. In this targeted approach (TMT-SRM), the selectivity of SRM is maintained while allowing the incorporation of an internal reference standard into the experiment. By monitoring 184 transitions, TMT-SRM resulted in the quantitation of 33 peptides representing 12 proteins. The acquired data corroborated the results obtained during the discovery phase. Furthermore, these data obtained by MS-based quantitation of peptides were independently confirmed by western blotting results, an orthogonal approach based on quantitation at the protein level.

Comparison of the nuclear proteomes of mammary epithelial cells at different stages of functional differentiation.

The progression of stem cells to proliferating progenitor cells and finally to a quiescent differentiated state is a hallmark of organ development. This process proceeds through distinct steps and is regulated through cell-cell interactions and by systemically and locally acting factors. We have established a cell culture system which recapitulates features of mammary gland development in vitro and allows the comparison of three characteristic differentiation stages. Cell fate decisions relating to proliferation and differentiation are dependent on the function of proteins in the nucleus. Therefore, we have applied proteomic approaches, including 1- and 2-DE coupled with MS and a gel-free system, called protein sequence tag technology (PST), to assess the changes in the nuclear protein composition during differentiation of mammary epithelial cells. We identified about 250 individual proteins which are present in the nucleus of proliferating and functionally differentiated mammary epithelial cells. We functionally categorised the differentially expressed proteins and identified a multitude of proteins that regulate gene expression at the transcriptional or post-transcriptional level. This analysis greatly enriches our global view of the dynamic changes of nuclear proteins during the development of mammary epithelial cells and suggests models for the control of differentiation-specific protein expression.

Protein sequence tags: a novel solution for comparative proteomics.

Comparative proteome profiling using stable isotope peptide labelling and mass spectrometry has emerged as a promising strategy. Here, we show the broad potential of our proprietary protein sequence tag (PST) technology. A special feature of PST is its ability to detect a wide variety of proteins including the pharmaceutically relevant membrane and nuclear proteins. This procedure addresses a similar number of proteins, compared to the multidimensional protein identification technology approach, but offers additionally a quantitative analysis with its recently developed quantitative PST version.

Characterization of low abundant membrane proteins using the protein sequence tag technology.

About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature. In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions. The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins. The identification of these N-terminal PST peptides is based on LC-MS/MS. The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment. The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins. More interestingly, among these membrane proteins 56% are predicted to be of low abundance. These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins.

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.