A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Direct radiolabeling of methotrexate and methotrexate micelles with Tc-99m using QbD approach.

The aim of the work presented in this manuscript was to radiolabel methotrexate and prepare radiolabeled methotrexate micelles, an antifolate drug with Tc-99m using QbD approach. The radiolabeling was executed using the experimental design and the radiolabeled drug was further encapsulated in micelles. The authors are of the view that the radiolabeled MTX could be used to target the folate receptor overexpressing cancers such as the kidney, colorectal, breast, brain etc thereby opening newer possibilities to the theranostic applications of the formed conjugate.

Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria.

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/13 C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC-MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.

Methods for Radiolabeling Nanoparticles (Part 3): Therapeutic Use.

Following previously published systematic reviews on the diagnostic use of nanoparticles (NPs), in this manuscript, we report published methods for radiolabeling nanoparticles with therapeutic alpha-emitting, beta-emitting, or Auger's electron-emitting isotopes. After analyzing 234 papers, we found that different methods were used with the same isotope and the same type of nanoparticle. The most common type of nanoparticles used are the PLGA and PAMAM nanoparticles, and the most commonly used therapeutic isotope is 177Lu. Regarding labeling methods, the direct encapsulation of the isotope resulted in the most reliable and reproducible technique. Radiolabeled nanoparticles show promising results in metastatic breast and lung cancer, although this field of research needs more clinical studies, mainly on the comparison of nanoparticles with chemotherapy.

Determining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.

In recent years, quantitative mass spectrometry-based interaction proteomics technology has proven very useful in identifying specific DNA-protein interactions using single pull-downs from crude lysates. Here, we applied a SILAC/TMT-based higher-order multiplexing approach to develop an interaction proteomics workflow called Protein-nucleic acid Affinity and Specificity quantification by MAss spectrometry in Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentration range of specific and control DNA baits are performed in SILAC-labeled nuclear extracts. MS1-based quantification to determine specific DNA-protein interactions is then combined with sequential TMT-based quantification of fragmented SILAC peptides, allowing the generation of Hill-like curves and determination of apparent binding affinities. We benchmarked PASMAN using the SP/KLF motif and further applied it to gain insights into two CGCG-containing consensus DNA motifs. These motifs are recognized by two BEN domain-containing proteins, BANP and BEND3, which we find to interact with these motifs with distinct affinities. Finally, we profiled the BEND3 proximal proteome, revealing the NuRD complex as the major BEND3 proximal protein complex in vivo. In summary, PASMAN represents, to our knowledge, the first higher-order multiplexing-based interaction proteomics method that can be used to decipher specific DNA-protein interactions and their apparent affinities in various biological and pathological contexts.

161Tb-DOTATOC Production Using a Fully Automated Disposable Cassette System: A First Step Toward the Introduction of 161Tb into the Clinic.

161Tb is an interesting radionuclide for application in the treatment of neuroendocrine neoplasms' small metastases and single cancer cells because of its conversion and Auger-electron emission. Tb has coordination chemistry similar to that of Lu; therefore, like 177Lu, it can stably radiolabel DOTATOC, one of the leading peptides used for the treatment of neuroendocrine neoplasms. However, 161Tb is a recently developed radionuclide that has not yet been specified for clinical use. Therefore, the aim of the current work was to characterize and specify 161Tb and to develop a protocol for the synthesis and quality control of 161Tb-DOTATOC with a fully automated process conforming to good-manufacturing-practice guidelines, in view of its clinical use. Methods: 161Tb, produced by neutron irradiation of 160Gd in high-flux reactors followed by radiochemical separation from its target material, was characterized regarding its radionuclidic purity, chemical purity, endotoxin level, and radiochemical purity (RCP) in analogy to what is described in the European Pharmacopoeia for no-carrier-added 177Lu. In addition, 161Tb was introduced into a fully automated cassette-module synthesis to produce 161Tb-DOTATOC, as used for 177Lu-DOTATOC. The quality and stability of the produced radiopharmaceutical in terms of identity, RCP, and ethanol and endotoxin content were assessed by means of high-performance liquid chromatography, gas chromatography, and an endotoxin test, respectively. Results: 161Tb produced under the described conditions showed, as the no-carrier-added 177Lu, a pH of 1-2, radionuclidic purity and RCP of more than 99.9%, and an endotoxin level below the permitted range (175 IU/mL), indicating its appropriate quality for clinical use. In addition, an efficient and robust procedure for the automated production and quality control of 161Tb-DOTATOC with clinically applicable specifications and activity levels, that is, 1.0-7.4 GBq in 20 mL, was developed. The radiopharmaceutical's quality control was also developed using chromatographic methods, which confirmed the product's stability (RCP ≥ 95%) over 24 h. Conclusion: The current study demonstrated that 161Tb has appropriate features for clinical use. The developed synthesis protocol guarantees high yields and safe preparation of injectable 161Tb-DOTATOC. The investigated approach could be translated to other DOTA-derivatized peptides; thus, 161Tb could be successfully applied in clinical practice for radionuclide therapy.

Quantification and Tracing of Stable Isotope into Cysteine and Glutathione.

The analysis of metabolic perturbation in biological samples is crucial to understand mechanisms of metabolic diseases. Here, we describe a protocol for quantitative stable isotope-labeled metabolite tracing of cysteine metabolism in cultured cells. This protocol relies on an extraction protocol to derivatize free thiols to prevent oxidation. In addition, the quantitative tracing of serine into multiple pathways, including the glutathione synthesis pathway, allows for the interrogation of cysteine and glutathione synthesis. This protocol provides a flexible framework that can be adapted to interrogate many metabolites and pathways of interest.

Diazo probe-based chemical isotope labeling assisted liquid chromatography-tandem mass spectrometry analysis for sensitive determination of amino acids in biofluids.

Amino acids are important biomolecules and contribute to essential biological processes. Liquid chromatography tandem mass spectrometry (LC-MS) now is a powerful tool for the analysis of amino acid metabolites; however, the structural similarity and polarity of amino acids can lead to the poor chromatographic retention and low detection sensitivities. In this study, we used a pair of light and heavy isotopomers of diazo probes, d0/d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA/d5 -2-DMBA) to label amino acids. The paired MS probes of 2-DMBA and d5 -2-DMBA carry diazo groups that can efficiently and specifically react with the carboxyl group on free amino acid metabolites under mild conditions. Benefiting from the transfer of the 2-DMBA/d5 -2-DMBA to carboxyl group on amino acids, the ionization efficiencies of amino acids presented great enhancement during LC-MS analysis. The results suggested that the detection sensitivities of 17 amino acids increased by 9-133-fold upon 2-DMBA labeling, and the obtained limits of detection (LODs) of amino acids on-column ranged from 0.011 fmol-0.057 fmol. With the application of the developed method, we successfully achieved the sensitive and accurate detection of the 17 amino acids in microliter level of serum sample. Moreover, the contents of most amino acids were different in the serum from normal and B16F10-tumour mice, demonstrating that endogenous amino acids may play important roles in the regulation of tumors development. This developed method of chemical labeling of amino acids with diazo probes assisted LC-MS analysis provides a potentially valuable tool to investigate the relationships between amino acids metabolism and diseases.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

Development of pseudo-targeted profiling of isotopic metabolomics using combined platform of high resolution mass spectrometry and triple quadrupole mass spectrometry with application of 13C6-glucose tracing in HepG2 cells.

Isotope tracing assisted metabolic analysis is becoming a unique tool to understand metabolic regulation in cell biology and biomedical research. Targeted mass spectrometry analysis based on selected reaction monitoring (SRM) has been widely applied in isotope tracing experiment with the advantages of high sensitivity and broad linearity. However, its application for new pathway discovery is largely restrained by molecular coverage. To overcome this limitation, we describe a strategy called pseudo-targeted profiling of isotopic metabolomics (PtPIM) to expand the analysis of isotope labeled metabolites beyond the limit of known pathways and chemical standards. Pseudo-targeted metabolomics was first established with ion transitions and retention times transformed from high resolution (orbitrap) mass spectrometry. Isotope labeled MRM transitions were then generated according to chemical formulas of fragments, which were derived from accurate ion masses acquired by HRMS. An in-house software "PseudoIsoMRM" was developed to simulate isotope labeled ion transitions in batch mode and correct the interference of natural isotopologues. This PtPIM strategy was successfully applied to study 13C6-glucose traced HepG2 cells. As 313 molecules determined as analysis targets, a total of 4104 ion transitions were simulated to monitor 13C labeled metabolites in positive-negative switching mode of QQQ mass spectrometer with minimum dwell time of 0.3 ms achieved. A total of 68 metabolites covering glycolysis, TCA cycle, nucleotide biosynthesis, one-carbon metabolism and related derivatives were found to be labeled (> 2%) in HepG2 cells. Active pentose phosphate pathway was observed with diverse labeling status of glycolysis intermediates. Meanwhile, our PtPIM strategy revealed that rotenone severely suppressed mitochondrial function e.g. oxidative phosphorylation and fatty acid beta-oxidation. In this case, anaerobic respiration became the major source of energy metabolism by producing abundant lactate. Conclusively, the simulation based PtPIM method demonstrates a strategy to broaden metabolite coverage in isotope tracing analysis independent of standard chemicals.

Genetic Code Expansion for Site-Specific Labeling of Antibodies with Radioisotopes.

Due to their target specificity, antibody-drug conjugates─monoclonal antibodies conjugated to a cytotoxic moiety─are efficient therapeutics that can kill malignant cells overexpressing a target gene. Linking an antibody with radioisotopes (radioimmunoconjugates) enables powerful diagnostics and/or closely related therapeutic applications, depending on the isotope. To generate site-specific radioimmunoconjugates, we utilized genetic code expansion and subsequent conjugation by inverse electron-demand Diels-Alder cycloaddition reactions. We show that, using this approach, site-specific labeling of trastuzumab with either zirconium-89 (89Zr) for diagnostics or lutetium-177 (177Lu) for therapeutics yields efficient radioimmunoconjugates. Positron emission tomography imaging revealed a high accumulation of site-specifically 89Zr-labeled trastuzumab in tumors after 24 h and low accumulation in other organs. The corresponding 177Lu-trastuzumab radioimmunoconjugates were comparably distributed in vivo.

Probing local changes to α-helical structures with 2D IR spectroscopy and isotope labeling.

α-Helical secondary structures impart specific mechanical and physiochemical properties to peptides and proteins, enabling them to perform a vast array of molecular tasks ranging from membrane insertion to molecular allostery. Loss of α-helical content in specific regions can inhibit native protein function or induce new, potentially toxic, biological activities. Thus, identifying specific residues that exhibit loss or gain of helicity is critical for understanding the molecular basis of function. Two-dimensional infrared (2D IR) spectroscopy coupled with isotope labeling is capable of capturing detailed structural changes in polypeptides. Yet, questions remain regarding the inherent sensitivity of isotope-labeled modes to local changes in α-helicity, such as terminal fraying; the origin of spectral shifts (hydrogen-bonding versus vibrational coupling); and the ability to definitively detect coupled isotopic signals in the presence of overlapping side chains. Here, we address each of these points individually by characterizing a short, model α-helix (DPAEAAKAAAGR-NH2) with 2D IR and isotope labeling. These results demonstrate that pairs of 13C18O probes placed three residues apart can detect subtle structural changes and variations along the length of the model peptide as the α-helicity is systematically tuned. Comparison of singly and doubly labeled peptides affirm that frequency shifts arise primarily from hydrogen-bonding, while vibrational coupling between paired isotopes leads to increased peak areas that can be clearly differentiated from underlying side-chain modes or uncoupled isotope labels not participating in helical structures. These results demonstrate that 2D IR in tandem with i,i+3 isotope-labeling schemes can capture residue-specific molecular interactions within a single turn of an α-helix.

The Dynamic and Crucial Role of the Arginine Methylproteome in Myoblast Cell Differentiation.

Protein arginine methylation is an extensive and functionally significant post-translational modification. However, little is known about its role in differentiation at the systems level. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics of whole proteome analysis in proliferating or five-day differentiated mouse C2C12 myoblasts, followed by high-resolution mass spectrometry, biochemical assays, and specific immunoprecipitation of mono- or dimethylated arginine peptides, we identified several protein families that were differentially methylated on arginine. Our study is the first to reveal global changes in the arginine mono- or dimethylation of proteins in proliferating myoblasts and differentiated myocytes and to identify enriched protein domains and novel short linear motifs (SLiMs). Our data may be crucial for dissecting the links between differentiation and cancer growth.

Liquid Chromatography-Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne.

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

Fast and Cost-Efficient 17 O-Isotopic Labeling of Carboxylic Groups in Biomolecules: From Free Amino Acids to Peptide Chains.

17 O NMR spectroscopy is a powerful technique, which can provide unique information regarding the structure and reactivity of biomolecules. However, the low natural abundance of 17 O (0.04 %) generally requires working with enriched samples, which are not easily accessible. Here, we present simple, fast and cost-efficient 17 O-enrichment strategies for amino acids and peptides by using mechanochemistry. First, five unprotected amino acids were enriched under ambient conditions, consuming only microliter amounts of costly labeled water, and producing pure molecules with enrichment levels up to ∼40 %, yields ∼60-85 %, and no loss of optical purity. Subsequently, 17 O-enriched Fmoc/tBu-protected amino acids were produced on a 1 g/day scale with high enrichment levels. Lastly, a site-selective 17 O-labeling of carboxylic functions in peptide side-chains was achieved for RGD and GRGDS peptides, with ∼28 % enrichment level. For all molecules, 17 O ssNMR spectra were recorded at 14.1 T in reasonable times, making this an important step forward for future NMR studies of biomolecules.

Heavy Methyl SILAC Metabolic Labeling of Human Cell Lines for High-Confidence Identification of R/K-Methylated Peptides by High-Resolution Mass Spectrometry.

Protein methylation is a widespread post-translational modification (PTM) involved in several important biological processes including, but not limited to, RNA splicing, signal transduction, translation, and DNA repair. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered today the most versatile and accurate technique to profile PTMs with high precision and proteome-wide depth; however, the identification of protein methylations by MS is still prone to high false discovery rates. In this chapter, we describe the heavy methyl SILAC metabolic labeling strategy that allows high-confidence identification of in vivo methyl-peptides by MS-based proteomics. We provide a general protocol that covers the steps of heavy methyl labeling of cultured cells, protein sample preparation, LC-MS/MS analysis, and downstream computational analysis of the acquired MS data.

Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods.

Proteins are integral to biological systems and functions. Identifying and quantifying proteins can therefore offer systems-wide insights into protein-protein interactions, cellular signaling, and biological pathway activity. The use of quantitative proteomics has become a method of choice for identifying and quantifying proteins in complex matrices. Proteomics allows researchers to survey hundreds to thousands of proteins in a less biased manner than classical antibody-based protein capture strategies. Typically, discovery approaches have used data-dependent acquisition (DDA) methods, but this approach suffers from stochasticity that compromises quantitation. Recent developments in data-independent acquisition (DIA) proteomics workflows enable proteomic profiling of thousands of samples with increased peak picking consistency making it an excellent candidate for discovering and assessing biomarkers in clinical samples. However, quantitation of peptides from DIA datasets is computationally intensive, and guidelines on how to establish DIA methods are lacking. Method development and optimization require novel tools to visualize and filter DIA datasets appropriately. Here, a protocol and novel script workflow for the optimization of quantitative DIA methods using stable isotope labeling of amino acids in culture (SILAC) are presented. This protocol includes steps for cell growth and labeling, peptide digestion and preparation, and optimization of quantitative DIA methods. In addition, important steps for (1) computational analysis to identify and quantify peptides, (2) data visualizations to identify the linear abundance ranges for all peptides in the sample, and (3) descriptions of how to find high confidence quantitation abundance thresholds are described herein.

SILAC-IodoTMT for Assessment of the Cellular Proteome and Its Redox Status.

Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).

Targeted Absolute Protein Quantification Using SILAC Internal Standard and Full-Length Protein Calibrators (TAQSI).

Mass spectrometry (MS)-based proteomics has been increasingly used for targeted absolute protein quantifications in both basic and clinical research. There is a great need to overcome some pitfalls of current MS-based targeted absolute protein quantification methods, such as high inter-assay variability and high cost associated with the use of synthesized isotopic peptides/proteins. Here we describe a targeted absolute protein quantification method utilizing SILAC internal standards and unlabeled full-length protein calibrators (TAQSI). The method has proven accurate, precise, reproducible, and cost-effective. Notably, the method is resistant to the variabilities caused by protein extraction and digestion. Moreover, it avoids measurement errors due to nonsynonymous mutations. This versatile method can be used for determining the absolute expressions of numerous proteins in various biological samples. As a proof-of-concept, this method was successfully applied to absolutely quantitate the protein expressions of carboxylesterase 1 (CES1) in human liver tissues.