Free Amino Terminus Research Articles

A Novel Non-Destructive Rapid Tool for Estimating Amino Acid Composition and Secondary Structures of Proteins in Solution.

Amino-acid protein composition plays an important role in biology, medicine, and nutrition. Here, a groundbreaking protein analysis technique that quickly estimates amino acid composition and secondary structure across various protein sizes, while maintaining their natural states is introduced and validated. This method combines multivariate statistics and the thermostable Raman interaction profiling (TRIP) technique, eliminating the need for complex preparations. In order to validate the approach, the Raman spectra are constructed of seven proteins of varying sizes by utilizing their amino acid frequencies and the Raman spectra of individual amino acids. These constructed spectra exhibit a close resemblance to the actual measured Raman spectra. Specific vibrational modes tied to free amino and carboxyl termini of the amino acids disappear as signals linked to secondary structures emerged under TRIP conditions. Furthermore, the technique is used inversely to successfully estimate amino acid compositions and secondary structures of unknown proteins across a range of sizes, achieving impressive accuracy ranging between 1.47% and 5.77% of root mean square errors (RMSE). These results extend the uses for TRIP beyond interaction profiling, to probe amino acid composition and structure.

Voltammetric determination of sulfamethoxazole using commercial screen-printed carbon electrodes

A differential pulse voltammetric (DPV) method using commercial screen-printed carbon electrodes (SPCE) is developed for a fast and cost-effective determination of the antibiotic sulfamethoxazole (SMX). Optimal measurements are carried out at pH 5.5 in acetate buffer and yield a detection limit of 15 µg/L and a linearity range of 50 – 600 µg/L with good repeatability (1.1%) and reproducibility (2.5%). The method is successfully applied to the analysis of a spiked tap water sample with very high reproducibility (0.4%) and good trueness (Recovery 95.2%).Additional considerations are made about the irreversible electrochemical oxidation of SMX, which is notoriously pH-dependent according to a process involving 2e- and 2H+, in agreement with several reported mechanisms in the literature. Besides, the analysis of other sulfonamide antibiotics is discussed, showing the key role of the free terminal amino group in oxidation, which makes almost undistinguishable voltammetric signals from different molecules sharing this moiety. In contrast, the acetylation of the mentioned group (e.g., in sulfonamide metabolites) drastically reduces the sensitivity of voltammetric measurements.

Inhibition of huntingtin aggregation by its N-terminal 17-residue peptide and its analogs

The N-terminal 17-residue stretch of huntingtin (httN17) folds into an amphipathic α-helix. The httN17–harboring polyQ peptides form oligomers that are mediated via the assembly of the httN17 α-helices. The oligomerization results in higher local concentration of the polyglutamine (polyQ) region, thereby facilitating amyloid formation. The httN17 co-assembles with the httN17-harbouring polyQ peptides, thereby reducing the local polyQ concentration, and consequently inhibiting aggregation. This study presents the aggregation inhibition of the exon I region of pathogenic huntingtin by httN17 and its analogs. The C-terminal amidation of httN17 is found to be essential for activity. The httN17 peptides with free amino terminus and the acetylated amino terminus possess comparable activity. The httN17 analog, wherein the Leu7 and Ala10 are substituted with 2-aminoisobutyric acid residues, exhibits significantly higher activity than the native httN17.

Application of different spectrofluorimetric approaches for quantitative determination of acetylsalicylic acid and omeprazole in recently approved pharmaceutical preparation and human plasma

Acetylsalicylic acid and omeprazole were recently formulated by the new FDA-approved drug Yosprala ® Tablets. This novel combination was prescribed to reduce the risk of myocardial infarction in patients who were at risk for developing peptic ulcer while taking acetylsalicylic acid. In the current work, two different high precision sensitive fluorescence spectroscopic methods were developed for quantitative analysis of the above drugs in pharmaceutical dosage form and spiked human plasma. Acetylsalicylic acid was quantitatively analyzed due to its unique native fluorescence nature. The fluorescence emission of acetylsalicylic acid was quantitatively determined at 404 nm after excitation at 296 nm without any interference from omeprazole. Omeprazole, which has a free terminal secondary amino group, reacted with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) by a nucleophilic substitution mechanism to form a highly fluorescent dark yellow fluorophore. Omeprazole was quantitatively analyzed by measuring the emission fluorescence intensity of the dark yellow fluorophore at 535 nm after excitation at 465 nm. Various parameters affecting the described methods were carefully checked and optimized. The calibration curves were found to be linear over the concentration range of 50–1600 ng/ml for acetylsalicylic and 30–2000 ng/ml for omeprazole. The proposed methods were successfully applied to the quantitative analysis of the two drugs in the pharmaceutical dosage form Yosprala ® and in spiked human plasma.

Application of different spectrofluorimetric methods for determination of lesinurad and allopurinol in pharmaceutical preparation and human plasma.

Lesinurad and allopurinol combination is newly FDA approved for treatment of patients suffering from hyperuricemia associated with uncontrolled gout. In the present work, two different highly sensitive, selective and accurate fluorescence spectroscopic methods were developed for quantitative analysis of lesinurad and allopurinol in their pharmaceutical dosage form without any tedious operation procedure. Lesinurad was quantitatively analyzed based on its unique native fluorescence nature. Lesinurad fluorescence emission was quantitatively determined at 343nm after excitation at 288nm without any interference from allopurinol. Allopurinol, has free terminal secondary amino group, reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBDCl) through nucleophilic substitution mechanism forming highly fluorescent dark yellow fluorophore. Allopurinol was quantitavely analyzed based on measurement the emission fluorescence intensity of the fluorescent dark yellow fluorophore at 535nm after excitation at 465nm. Different parameters which affect the described methods of the studied drugs were carefully checked and optimized. Calibration graphs were found to be linear over the concentration range of 0.25-4.0μg/mL for lesinurad and 0.2-20μg/mL for allopurinol. The proposed methods were successfully applied for the quantitative analysis of the two drugs in Duzallo® pharmaceutical dosage form and spiked human plasma.

Antiproliferative S-Trityl-l-Cysteine -Derived Compounds as SIRT2 Inhibitors: Repurposing and Solubility Enhancement.

S-trityl-l-cysteine (STLC) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S-trityl-l-histidine scaffold. Herein, we propose new STLC-derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 μM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC, presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.

Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity.

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

Crystal structure analysis of dipeptidyl amino peptidase from P. mexicana WO24.

Dipeptidyl aminopeptidase (DAP or DPP, EC 3.4.14) catalyses the removal of dipeptides from the amino termini of peptides and proteins. In microorganisms, we have reported the identification, purification, and characterization of DAP BI, DAP BII, DAP BIII, and DAP IV (bacterial DPP4), POP fromPseudoxanthomonas mexicanaWO24, and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family and they are classified into the clan SC, family S9 in the MEROPS database. On the basis of the enzymological data we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for the DAP IV. The DAP IV liberates dipeptides from the free amino terminus and has a specificity for both proline and hydroxyproline residues in the penultimate position of peptides. Here, we report the first structure of the bacterial DPP IV (P. mexicanaWO24 DAP IV) complexed with an inhibitor at 2.2 Å resolution. The subunit structure is composed of two domains, the N-terminal eight-bladed β-propeller domain and the C-terminal alpha/beta/alpha sandwich catalytic domain. These structural features are conserved with clan SC S9 family. However, the N-terminal domain contains a unique helix region that extends over the active site acting as a lid, gating substrate or product access. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique helix region is conserved in some kind of bacterial DPP4s except for mammalian DPP4s and some bacterial DPP4s. Some asaccharolytic and anaerobic bacteria can be used protein or peptides as an energy source. Therefore, these bacteria secrete many types of proteases and peptidases. Especially, the elucidation of degradation mechanisms of collagen, including proline and hydroxyproline, are very important from the point of view of host tissue breakdown in pathogens. Our findings suggest that different ligand recognition mechanisms from the bacterial DPP IV to mammalian DPP4 raise the possibility of an antimicrobial development targeting DPP IV from bacteria.

A spectroscopic and electrochemical investigation of the oxidation pathway of glycyl-d,l-methionine and its N-acetyl derivative induced by gold(III)

The 1H nuclear magnetic resonance spectroscopy was applied to study the reaction of the dipeptide glycyl-d,l-methionine (H-Gly-d,l-Met-OH) and its N-acetylated derivative (Ac-Gly-d,l-Met-OH) with hydrogen tetrachloridoaurate(III) (H[AuCl4]). The corresponding peptide and [AuCl4]– were reacted in 1:1, 2:1, and 3:1 molar ratios, and all reactions were performed at pD 2.45 in 0.01 M DCl as solvent and at 25°C. It was found that the first step of these reactions is coordination of Au(III) to the thioether sulfur atom with formation of the gold(III)-peptide complex [AuCl3(R-Gly-Met-OH-S)] (R=H or Ac). This intermediate gold(III) complex further reacts with an additional methionine residue to generate the R-Gly-Met-OH chlorosulfonium cation as the second intermediate product, which readily undergoes hydrolysis to give the corresponding sulfoxide. The oxidation of the methionine residue in the reaction between H-Gly-d,l-Met-OH and [AuCl4]– was five times faster (k 2 = 0.363 ± 0.074 M-1s-1) in comparison to the same process with N-acetylated derivative of this peptide (k 2 = 0.074 ± 0.007 M-1s-1). The difference in the oxidation rates between these two peptides can be attributed to the free terminal amino group of H-Gly-d,l-Met-OH dipeptide. The mechanism of this redox process is discussed and, for its clarification, the reaction of the H-Gly-d,l-Met-OH dipeptide with [AuCl4]– was additionally investigated by UV-Vis and cyclic voltammetry techniques. From these measurements, it was shown that the [AuCl2]– complex under these experimental conditions has a strong tendency to disproportionate, forming [AuCl4]– and metallic gold. This study contributes to a better understanding of the mechanism of the Au(III)-induced oxidation of methionine and methionine-containing peptides in relation to the severe toxicity of anti-arthritic and anticancer gold-based drugs.

Peptide-tags for enhanced DNA microarray performance

DNA microarrays are powerful tools for gene expression analysis and genotyping studies in research and diagnostic applications. A high sensitivity and short time-to-result are prerequisites for their practical application in the clinic. The hybridization efficiency of DNA microarrays depends on the probe density and the probe orientation and thus their accessibility for target molecules. In order to find an optimal probe immobilization procedure a set of different oligonucleotide modifications was tested on epoxy silane functionalized glass slides. It was found that histidine-tagged oligonucleotides resulted in the highest amount of bound probe and by far the best hybridization efficiencies. The detection limit obtained with histidine-tagged probes was up to two orders of magnitude lower compared to commonly used probe modifications. In order to further investigate the binding mechanism of histidine-tags towards functionalized glass substrates a set of different peptide-tags with and without free terminal amino-groups and with different amino acid compositions was tested. The results indicate an impact of the terminal amino group on the covalent surface binding and of aromatic amino acid residues on the enhanced hybridisation efficiency.

Structural Characterization of a High Affinity Mononuclear Site in the Copper(II)-α-Synuclein Complex

Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

Synthesis and Biological Characterization of Novel Charge-Deficient Spermine Analogues

Biogenic polyamines, spermidine and spermine, are positively charged at physiological pH. They are present in all cells and essential for their growth and viability. Here we synthesized three novel derivatives of the isosteric charge-deficient spermine analogue 1,12-diamino-3,6,9-triazadodecane (SpmTrien, 5a) that are N(1)-Ac-SpmTrien (5c), N(12)-Ac-SpmTrien (5b), and N(1),N(12)-diethyl-1,12-diamino-3,6,9-triazadodecane (N(1),N(12)-Et(2)-SpmTrien, 5d). 5a and 5d readily accumulated in DU145 cells at the same concentration range as natural polyamines and moderately competed for the uptake with putrescine (1) but not with spermine (4a) or spermidine (2). 5a efficiently down-regulated ornithine decarboxylase and decreased polyamine levels, while 5d proved to be inefficient, compared with N(1),N(11)-diethylnorspermine (6). None of the tested analogues were substrates for human recombinant spermine oxidase, but those having free aminoterminus, including 1,8-diamino-3,6-diazaoctane (Trien, 3a), were acetylated by mouse recombinant spermidine/spermine N(1)-acetyltransferase. 5a was acetylated to 5c and 5b, and the latter was further metabolized by acetylpolyamine oxidase to 3a, a drug used to treat Wilson's disease. Thus, 5a is a bioactive precursor of 3a with enhanced bioavailability.

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA(6)). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA(6) with a t((1/2)) of approximately 3 min at 37 degrees C. Maximum activation of degradation of the N-end rule substrate, LR-GFP(Venus), occurs with a single ClpS bound per ClpA(6); one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA(6), ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA(6). Limiting ClpS-mediated substrate delivery to one per ClpA(6) avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.

Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mIC(c12)) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C(18) RP-HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mIC(c12) cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll-like receptors (TLR). Whereas intact colostrum generally reduced TLR-mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum.

Extending the use of “Inverted Chirality Columns Approach” for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative

The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs.

Biocompatible and bioactive gum Arabic coated iron oxide magnetic nanoparticles

The surface modification of iron oxide magnetic nanoparticles (MNPs) with gum Arabic (GA) via adsorption and covalent coupling was studied. The adsorption of GA was assessed during MNP chemical synthesis by the co-precipitation method (MNP_GA), and after MNP synthesis on both bare magnetite and MNP_GA. The covalent immobilization of GA at the surface of aldehyde-activated (MNP_GA APTES) or aminated MNPs (MNP_GA EDC) was achieved through free terminal amino and carboxylate groups from GA. The presence of GA at the surface of the MNPs was confirmed by FTIR and by the quantification of GA by the bicinchoninic acid test. Results indicated that the maximum of GA coating was obtained for the covalent coupling of GA through its free carboxylate groups (MNP_GA EDC), yielding a maximum of 1.8 g of GA bound/g of dried particles. The hydrodynamic diameter of MNPs modified with GA after synthesis resulted in the lowest values, in opposition to the MNPs co-precipitated with GA which presented the tendency to form larger aggregates of up to 1 μm. The zeta potentials indicate the existence of negatively charged surfaces before and after GA coating. The potential of the GA coated MNPs for further biomolecule attachment was assessed through anchorage of a model antibody to aldehyde-functionalized MNP_GA and its subsequent detection with an FITC labeled anti-antibody.

Cover Picture: Proteolysis of Peptide Dendrimers (ChemBioChem 9/2009)

AbstractThe cover picture shows the discovery of proteolysis sites within peptide dendrimers. On p. 1527 ff. of this issue, J.‐L. Reymond et al. explain how proteolytic cleavage within the branches of these molecular trees was found to be possible by proteases such as trypsin (Trp) or chymotrypsin (Chy) when the correct amino acid (Arg for Trp, Phe for Chy) was placed at the cleavage site. Protease‐reactive dendrimers were identified by screening a one‐bead–one‐compound combinatorial library of dendrimers (top left) using an on‐bead proteolysis assay (red indicates the presence of free amino termini after proteolysis). It was also possible to hide proteolysis sites from the proteases by using a higher degree of branching in the dendrimers (not shown). A space‐filling rendering of a peptide dendrimer and its cleavage products upon trypsin cleavage is shown. Cover design and realization by Louise A. Reymond and Viviana S. Fluxa.magnified image

Protein Modification by Acrolein: Formation and Stability of Cysteine Adducts

The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intramolecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed, but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues.

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.

Mass spectrometric identification of pyroglutamic acid in peptides following selective hydrolysis

The formation of a pyroglutamyl residue by enzymatic cyclization of an amino terminus glutamine residue is a common posttranslational modification [1–3]. Several biologically active peptides, most notably the thyrotropinreleasing hormone $(TRH)^1$ and gonadotropin-releasing hormone (GnRH), were among the first examples where the N-terminal modification was characterized. The widespread occurrence of glutaminyl cyclase in both plants and animals has ensured that the number of naturally occurring pyroglutamyl peptides is rapidly growing. A recent report established formation of pyroglutamic acid (Z) from N-terminal glutamic acid in recombinant monoclonal antibodies [4]. An N-terminus pyroglutamic acid residue impedes Edman sequencing, necessitating the use of a specific enzyme, pyroglutamate aminopeptidase, to liberate a peptide with a free amino terminus, which is then amenable to automated sequence analysis [5]. During the course of studies aimed at characterizing the peptide components of Conus venom, we encountered several examples of pyroglutamyl peptides. We describe here the serendipitous finding of mild hydrolysis conditions, which permit opening of the pyroglutamyl ring, without significant cleavage of primary and secondary amide bonds.