Methionine-containing Peptides Research Articles

Stability of a Multiresponsive Sulfonium Vinyl Sulfide Linker toward Nucleophilic/Radical Thiols, Reactive Nitrogen Species, and in Cells under Pro-inflammatory Stimulation.

Chemical linkages that respond to biological stimuli are important for many pharmaceutical and biotechnological applications, making it relevant to explore new variants with different responsivity profiles. This work explores the responsiveness of a TAT peptide-based sulfonium vinyl sulfide probe that responds to nucleophilic thiols, radical thiol species (RTS), and reactive nitrogen species (RNS). Under model conditions, response to nucleophilic thiols was very slow (hours/days), though fast with down to molar equivalents of either RTS or RNS (minutes). These reactions led to the traceless release of a methionine-containing peptide in the first two cases and to a hydroxy nitration adduct in the third case. Despite the sensitive nature of the probe, it remained stable for at least ∼2 h in the presence of cells during TAT-mediated trafficking, even under pro-inflammatory stimulation. The thiol-responsiveness is intermediate to that observed for disulfide linkers and conventional cysteine-maleimide linkers, presenting opportunities for biotechnological applications.

ROS-Responsive Methionine-Containing Amphiphilic Peptides Impart Enzyme-Triggered Phase Transition and Antioxidant Cell Protection.

Reactive oxygen species (ROS) are produced by cellular activities, such as metabolism and immune response, and play important roles in cell signaling and homeostasis. However, overproduced ROS causes irreversible damage to nucleic acids and membrane lipids, supporting genetic mutations and enhancing the effects of aging. Cells defend themselves against ROS using antioxidant systems based on redox-active sulfur and transition metals. Inspired by such biological redox-responsive systems, we developed methionine-containing self-assembling peptides. The Met-containing peptides formed hydrogels that underwent a gel-to-sol phase transition upon oxidation by H2O2, and the sensitivity of the peptides to the oxidant increased as the number of Met residues increased. The peptide containing three Met residues, the largest number of Met residues in our series of designed peptides, showed the highest sensitivity to oxidation and detoxification to protect cells from ROS damage. In addition, this peptide underwent a phase transition in response to H2O2 produced by an oxidizing enzyme. This study demonstrates the design of a supramolecular biomaterial that is responsive to enzymatically generated ROS and can protect cells against oxidative stress.

Host-guest binding between cucurbit[8]uril and amphiphilic peptides achieved tunable supramolecular aggregates for cancer diagnosis.

The manipulation of biocompatible supramolecular nanostructures at subcellular and cellular levels has become one of the increasingly significant topics but remains a formidable challenge in chemical and biological science. In this work, a controllable supramolecular aggregate based on host-guest competitive binding is elaborately constructed using cucurbit[8]uril, methionine-containing amphiphilic peptide, and perylene diimide, displaying in situ oxidation-driven macrocycle-confined fluorescence enhancement for cell imaging and morphological reconstruction for cancer cell death. The experimental results demonstrate that cucurbit[8]uril possesses a high binding affinity with the methionine peptide, while this value sharply decreases after the methionine residue is oxidized to sulfoxide or sulfone. Therefore, perylene diimide can be competitively included by cucurbit[8]uril in the co-assemblies, eventually resulting in a 10-fold fluorescence enhancement and the conversion of topological morphology from nano-sized particles to micron-sized sheets. Moreover, the obtained ternary assemblies can be oxidized by endogenous reactive oxygen species in cancer cells, thus not only providing enhanced fluorescence for cell imaging, but also leading to endoplasmic reticulum dysfunction and significant cell death. Therefore, the controllable and oxidation-responsive morphological transformation based on the host-guest competitive binding in biological media can be viewed as a feasible means for efficient disease theragnosis.

Methionine-Containing Peptides: Avoiding Secondary Reactions in the Final Global Deprotection.

The solid-phase synthesis of Met-containing peptides using a fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) protection scheme is inevitably accompanied by two stubborn side reactions, namely, oxidation and S-alkylation (tert-butylation), which result in the formation of Met(O) and sulfonium salt impurities of the target peptide, respectively. These two reactions are acid-catalyzed, and they occur during the final trifluoroacetic (TFA)-based acidolytic cleavage step. Herein, we developed two new cleavage solutions that eradicate the oxidation and reduce S-alkylation. TFA-anisole-trimethylsilyl chloride (TMSCl)-Me2S-triisopropylsilane (TIS) containing 1 mg of triphenyl phosphine per mL of solution was the optimal mixture for Cys-containing peptides, while for the remaining peptides, TIS was not required. Both cleavage solutions proved to be excellent when sensitive amino acids such as Cys and Trp were involved. TMSCl did not affect either of these sensitive amino acids. Reversing the sulfonium salt to free Met-containing peptide was achieved by heating the peptide at 40 °C for 24 h using 5% acetic acid.

Consecutive Alkylation, "Click", and "Clip" Reactions for the Traceless Methionine-Based Conjugation and Release of Methionine-Containing Peptides.

"Click" reactions have revolutionized research in many areas of science. However, a disadvantage of the high stability of the Click product is that identifying simple treatments for cleanly dissociating the latter under the same guiding principles, i.e., a "Clip" reaction, remains a challenge. This study demonstrates that electron-deficient alkynes, conveniently installed on methionine residues, can participate in well-known Click (nucleophilic thiol-allene addition) and subsequent Clip reactions (radical thiol-ene addition). To illustrate this concept, a variety of bioconjugates (peptide-peptide; peptide-fluorophore; peptide-polymer; and peptide-protein) were prepared. Interestingly, the Clip reaction of these bioconjugates releases the original peptides concurrent with regeneration of their unmodified methionine residue, in minutes. Moreover, the conjugates demonstrate substantial stability toward endogenous levels of reactive species in bacteria, illustrating the potential for this chemistry in the biosciences. The reaction conditions employed in the Click and Clip steps are compatible with the preservation of the integrity of biomolecules/fluorophores and involve readily accessible reagents and the natural functional groups on peptides/proteins.

Dehydromethionine is a common product of methionine oxidation by singlet molecular oxygen and hypohalous acids

Methionine is one of the main targets for biological oxidants. Its reaction with the majority of oxidants generates only methionine sulfoxide. However, when N-terminal methionine reacts with hypohalous acids (HOCl and HOBr) or singlet molecular oxygen (1O2), it can also generate a cyclic product called dehydromethionine (DHM). Previously, DHM was suggested as a biomarker of oxidative stress induced by hypohalous acids. However, DHM can also be generated by 1O2 –oxidation of methionine, and the contribution of this pathway of DHM formation in a context of a site-specific redox imbalance in an organism is unknown. In this work, a through comparison of the reactions of hypohalous acids and 1O2 with methionine, either free or inserted in peptides and proteins was undertaken. In addition, we performed methionine photooxidation in heavy water (H218O) to determine the influence of the pH in the mechanism of DHM formation.We showed that for free methionine, or methionine-containing peptides, the yields of DHM formation in the reactions with 1O2 were close to those achieved by HOBr oxidation, but much higher than the yields obtained with HOCl as the oxidant. This was true for all pH tested (5, 7.4, and 9). Interestingly, for the protein ubiquitin, DHM yields after reaction with 1O2 were higher than those obtained with both hypohalous acids. Our results indicate that 1O2 may also be an important source of DHM in biological systems.

Discovery of the Xenon-Protein Interactome Using Large-Scale Measurements of Protein Folding and Stability.

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low-throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein target discovery. Here, we report the application of stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5000 methionine-containing peptides and over 5000 semi-tryptic peptides, mapping to ∼1500 and ∼950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified are tied to previously established modes of action related to xenon's anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein-gas interactions in complex biological mixtures, such as cell lysates.

Detecting Differences in Prion Protein Conformation by Quantifying Methionine Oxidation.

A prion’s pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion’s conformation and to distinguish among prion strains, which is an important component of food safety.

A peptide-crosslinking approach identifies HSPA8 and PFKL as selective interactors of an actin-derived peptide containing reduced and oxidized methionine.

The oxidation of methionine to methionine sulfoxide occurs under conditions of cellular oxidative stress, and modulates the function of a diverse array of proteins. Enzymatic systems that install and reverse the methionine sulfoxide modifications have been characterized, however, little is known about potential readers of this oxidative modification. Here, we apply a peptide-crosslinking approach to identify proteins that are able to differentially interact with reduced and oxidized methionine-containing peptides. Specifically, we generated a photo-crosslinking peptide derived from actin, which contains two sites of methionine oxidation, M44 and M47. Our proteomic studies identified heat shock proteins, including HSPA8, as selective for the reduced methionine-containing peptide, whereas the phosphofructokinase isoform, PFKL, preferentially interacts with the oxidized form. We then demonstrate that the favored interaction of PFKL with oxidized methionine is also observed in the full-length actin protein, suggesting a role of methionine oxidation in regulating the actin-PFKL interaction in cells. Our studies demonstrate the potential to identify proteins that can differentiate between reduced and oxidized methionine and thereby mediate downstream protein functions under conditions of oxidative stress. Furthermore, given that numerous sites of methionine oxidation have now been identified, these studies set the stage to identify putative readers of methionine oxidation on other protein targets.

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

Formation of peptide disulfide bonds through a trans-dibromido-Pt(IV) complex oxidation reaction: Kinetic and mechanistic analyses

A trans-dibromido-Pt(IV) complex [PtBr2(en)2]Br2 was synthesized and evaluated for the synthesis of peptide disulfide bonds in this work. The reactions between the Pt(IV) complex and dicysteine-containing peptides were carried out in various aqueous solutions. As a result, excellent yields with fast reaction rates were achieved. Methionine residue was intact when the Pt(IV) complex reacted with a dicysteine and methionine-containing peptide. 3,6-Dioxa-1,8-octanedithiol (DODT) was selected as a model compound of the dicysteine-containing peptide. Kinetic studies for the reaction between the Pt(IV) complex and DODT were performed in different pH solutions. It is first-order both in [Pt(IV)] and in [DODT]. A reaction mechanism was proposed accordingly. The kinetic and mechanistic results demonstrated that the reaction rate for the formation of peptide disulfide bond via the Pt(IV) oxidation is increased with the increase of pH of the reaction medium. The protolytic constants of the two thiol groups in peptide also play a critical role in reaction rate. On the other hand, kinetic studies demonstrated that the Pt(IV) complex trans-[PtBr2(en)2]2+ can be used in an acidic medium for the purpose of synthesis of disulfide bonds.

Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring.

Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor A (VEGF-A) and is used to treat various cancers and recently COVID-19. The dosage recommendations for bevacizumab are determined on the basis of body weight, and the drug is administered after defined time intervals, when it is presumed to still be above its minimum effective serum concentration. Interindividual and disease-stage-related variations in bevacizumab catabolism, however, can affect the proper dosing of patients, resulting in plasma concentrations which may not be within the optimal therapeutic window for the drug. Therapeutic drug monitoring (TDM) enables the assessment of patients' serum concentrations and allows personalized dosing which has the potential to improve efficacy and reduce side effects. While TMD is often performed using ligand-based assays, mass spectrometry (MS)-based TDM offers improved specificity. Here, we present a robust multiple reaction monitoring (MRM)-MS-based TDM method for the precise quantification of bevacizumab plasma concentrations, based on the controlled oxidation of the methionine-containing peptide, STAYLQMNSLR. The assay shows good linearity (r 2 = 0.9951), robustness, and precision (CVs < 20%) for the quantification of bevacizumab, with a lower limit of quantification (S/N > 10) of 1.8 μg/mL of plasma, without the need for enrichment and requiring less than 1 μL of plasma and less than 6 h from sampling to result.

Characterization of SETD3 methyltransferase–mediated protein methionine methylation

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310 Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.

Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS)

Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called ‘surrogate peptides’, that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 μL of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (H2O2) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using H2O2 to oxidize methionine residues and the enzyme catalase to quench excess H2O2. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins.

Synthetic integrin-binding immune stimulators target cancer cells and prevent tumor formation

Immuno-oncology approaches mainly utilize monoclonal antibodies or protein-based scaffolds that bind with high affinity to cancer cells and can generate an immune response. Peptides can also bind with high affinity to cancer cells and are intermediate in size between antibodies and small molecules. They are also synthetically accessible and therefore easily modified to optimize their stability, binding affinity and selectivity. Here we describe the design of immune system engagers (ISErs), a novel class of synthetic peptide-based compounds that bind specifically to cancer cells and stimulate the immune system. A prototype, Y9, targets integrin α3, which is overexpressed on several cancer cells, and activates the immune system via a formyl methionine-containing effector peptide. Injection of Y9 leads to immune cell infiltration into tissue and prevents tumor formation in a guinea pig model. The anti-tumor activity and synthetic accessibility of Y9 illustrate that ISErs could be applied to a wide variety of targets and diseases.

Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents.

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was “QAMXXMM-E/G/M-S/G-A/G/F-XG”. We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.

Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

Highly Sensitive Antibody-Free μLC–MS/MS Quantification of Rhtrail in Serum

We describe an antibody-free approach to quantify rhTRAIL(WT) (wild-type) and its closely related death receptor 4 selective variant rhTRAIL(4C7) in human and murine serum by multiplex LC-MS/MS on a microfluidics interface. Enrichment of rhTRAIL was performed by strong cation-exchange (SCX) followed by immobilized metal affinity (IMAC) solid-phase extraction. This was followed by trypsin digestion and using methionine-containing signature peptides after fully oxidizing the methionine residue with 0.25% (w/w) hydrogen peroxide. Absolute quantification was reaching down to 0.5 ng/ml for rhTRAIL(WT) (8.5 pM) and 2 ng/ml for rhTRAIL(4C7) (34 pM) in 100 μl human serum. To support preclinical studies in mice, the analysis was optimized further, for a sample volume of 20 μl murine serum.