ABSTRACT
T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as â¼0.5â¯nM for the four analytes, with quantification extended to 20â¯nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8â¯nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.
Subject(s)
Autoimmune Diseases , Immunoconjugates , Humans , CD28 Antigens/metabolism , Antigens, CD/metabolism , B7-2 Antigen , Chromatography, Liquid , Liquid Chromatography-Mass Spectrometry , Tandem Mass Spectrometry , B7-1 Antigen/metabolism , AbataceptABSTRACT
Elevating soluble CD80 (sCD80) in human serum is a natural response to autoimmune diseases such as rheumatoid arthritis (RA). The level of sCD80 is associated with RA development and prognosis; therefore, it is potentially used as a biomarker. sCD80 is commonly measured in human serum using immunoassays (e.g., ELISA) with multiple drawbacks, mainly cross-reactivity. Aptamer-based biosensors (aptasensors) development for quantifying and detecting different biological molecules demonstrates applicability in next-generation medicine and biomarker detection. Herein, we selected a specific aptamer for sCD80 by conventional in-vitro selection process (SELEX) with the high-affinity aptamer (Kd = 47.69 nM). A sensitive aptasensor, for the first time, was developed on a screen-printed gold electrode (AuSPE) platform compatible with easy-to-use label-free electrochemical impedance spectroscopy. The immobilization of the aptamer on the gold surface and the presence of sCD80 in a complex with the aptamer were characterized by photo-induced force microscopy, which revealed the uniform assembly of the aptamer monolayer and the distribution of sCD80 on the electrode surface. The developed aptasensor showed a linear performance (0.025-10.0 nM of protein) with a detection limit of 8.0 pM. Furthermore, the aptasensor was tested in a biological matrix, where a linear signal was observed for the increased amount of spiked sCD80 (R2 = 0.9887). The recovery of the spiked amounts ranged from 105 to 125% with coefficient of variation (CV%) <7%, which supported the applicability of this sensor in detecting sCD80 for diagnosis.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Humans , Biosensing Techniques/methods , Limit of Detection , Electrochemical Techniques/methods , Aptamers, Nucleotide/chemistry , Cell Adhesion Molecules , Gold/chemistry , Biomarkers , ElectrodesABSTRACT
In aquatic ecosystems, microalgae are exposed to light fluctuations at different frequencies due to daily and seasonal changes. Although concentrations of herbicides are lower in Arctic than in temperate regions, atrazine and simazine, are increasingly found in northern aquatic systems because of long-distance aerial dispersal of widespread applications in the south and antifouling biocides used on ships. The toxic effects of atrazine on temperate microalgae are well documented, but very little is known about their effects on Arctic marine microalgae in relation to their temperate counterparts after light adaptation to variable light intensities. We therefore investigated the impacts of atrazine and simazine on photosynthetic activity, PSII energy fluxes, pigment content, photoprotective ability (NPQ), and reactive oxygen species (ROS) content under three light intensities. The goal was to better understand differences in physiological responses to light fluctuations between Arctic and temperate microalgae and to determine how these different characteristics affect their responses to herbicides. The Arctic diatom Chaetoceros showed stronger light adaptation capacity than the Arctic green algae Micromonas. Atrazine and simazine inhibited the growth and photosynthetic electron transport, affected the pigment content, and disturbed the energy balance between light absorption and utilization. As a result, during high light adaptation and in the presence of herbicides, photoprotective pigments were synthesized and NPQ was highly activated. Nevertheless, these protective responses were insufficient to prevent oxidative damage caused by herbicides in both species from both regions, but at different extent depending on the species. Our study demonstrates that light is important in regulating herbicide toxicity in both Arctic and temperate microalgal strains. Moreover, eco-physiological differences in light responses are likely to support changes in the algal community, especially as the Arctic ocean becomes more polluted and bright with continued human impacts.
Subject(s)
Atrazine , Chlorophyta , Herbicides , Microalgae , Water Pollutants, Chemical , Humans , Herbicides/toxicity , Simazine/pharmacology , Ecosystem , Water Pollutants, Chemical/toxicityABSTRACT
Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.
Subject(s)
Amine Oxidase (Copper-Containing) , Amino Acids, Diamino , Lathyrus , Chromatography, Liquid/methods , Amine Oxidase (Copper-Containing)/metabolism , Tandem Mass Spectrometry , Amino Acids, Diamino/analysis , Amino Acids, Diamino/chemistry , Amino Acids, Diamino/metabolismABSTRACT
Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.
Subject(s)
Bile Acids and Salts , Chemical and Drug Induced Liver Injury , Rats , Animals , Acetaminophen/adverse effects , Glucuronides , Mass Spectrometry/methodsABSTRACT
Polar ecosystems play an important role in global primary production. Microalgae have adaptations that enable them to live under low temperature environments where irradiance and day length change drastically. Their adaptations, leading to different ecophysiological characteristics relative to temperate species, could also alter their sensitivity to pollutants such as pesticides. This study's objective was to understand how different ecophysiological characteristics influence the response of Arctic phytoplankton to pesticides in relation to the responses of their temperate counterparts. Ecophysiological endpoints were related to growth, cell biovolume, pigment content, photosynthetic activity, photoprotective mechanisms (NPQ, antioxidant enzyme activities), and reactive oxygen species (ROS) content. The Arctic species Micromonas polaris was more resistant to atrazine and simazine than its temperate counterpart Micromonas bravo. However, the other Arctic species Chaetoceros neogracilis was more sensitive to these herbicides than its temperate counterpart Chaetoceros neogracile. With respect to two other pesticide toxicity, both temperate microalgae were more sensitive to trifluralin, while Arctic microalgae were more sensitive to chlorpyrifos (insecticide). All differences could be ascribed to differences in the eco-physiological features of the two microalgal groups, which can be explained by cell size, pigment content, ROS content and protective mechanisms (NPQ and antioxidant enzymes).
Subject(s)
Chlorophyta , Microalgae , Pesticides , Water Pollutants, Chemical , Pesticides/toxicity , Pesticides/analysis , Reactive Oxygen Species , Antioxidants , Ecosystem , Water Pollutants, Chemical/toxicityABSTRACT
Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.
Subject(s)
Breast Neoplasms , Zebrafish , Humans , Female , Animals , Mice , Pilot Projects , HeterograftsABSTRACT
Dyskinesia remains an unmet need in Parkinson's disease (PD). We have previously demonstrated that glycine transporter 1 (GlyT1) inhibition with ALX-5407 reduces dyskinesia and slightly improves parkinsonism in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset. Here, we sought to determine the effect of bitopertin, a clinically-tested GlyT1 inhibitor, on parkinsonism and dyskinesia in the 6-hydroxydopamine (6-OHDA)-lesioned rat. To do so, we assessed the effect of bitopertin on parkinsonism as monotherapy and as adjunct to a low dose of L-3,4-dihydroxyphenylalanine (L-DOPA). We then assessed the efficacy of bitopertin on dyskinesia in the context of acute challenge and chronic administration studies. Lastly, we evaluated whether de novo treatment with bitopertin, started concurrently with L-DOPA, would diminish the development of dyskinesia. We discovered that bitopertin (0.3 mg/kg), when administered alone, reduced the severity of parkinsonism by 35% (P < 0.01). As adjunct to a low dose of L-DOPA, bitopertin (3 mg/kg) enhanced the anti-parkinsonian effect of L-DOPA by 36% (P < 0.05). Moreover, the acute addition of bitopertin (0.03 mg/kg) to L-DOPA reduced dyskinesia by 27% (P < 0.001), and there was no tolerance to the anti-dyskinetic benefit after 4 weeks of daily administration. Lastly, bitopertin (0.03 mg/kg) started concurrently with L-DOPA, also attenuated the development of dyskinesia, by 33% (P < 0.01), when compared to L-DOPA alone. Our results suggest that GlyT1 inhibition may simultaneously reduce parkinsonism and L-DOPA-induced dyskinesia and represents a novel approach to treat, possibly prevent, motor complications in PD.
Subject(s)
Dyskinesia, Drug-Induced , Parkinson Disease , Parkinsonian Disorders , Animals , Antiparkinson Agents/adverse effects , Dyskinesia, Drug-Induced/drug therapy , Dyskinesia, Drug-Induced/etiology , Glycine Plasma Membrane Transport Proteins , Levodopa/pharmacology , Oxidopamine , Parkinson Disease/drug therapy , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/drug therapy , Piperazines , Rats , SulfonesABSTRACT
Copper (Cu) is a redox-active transition element critical to various metabolic processes. These functions are accomplished in tandem with Cu-binding ligands, mainly proteins. The main goal of this work was to understand the mechanisms that govern the intracellular fate of Cu in the freshwater green alga, Chlamydomonas reinhardtii, and more specifically to understand the mechanisms underlying Cu detoxification by algal cells in low-Fe conditions. We show that Cu accumulation was up to 51-fold greater for algae exposed to Cu in low-Fe medium as compared to the replete-Fe growth medium. Using the stable isotope 65Cu as a tracer, we studied the subcellular distribution of Cu within the various cell compartments of C. reinhardtii. These data were coupled with metallomic and proteomic approaches to identify potential Cu-binding ligands in the heat-stable proteins and peptides fraction of the cytosol. Cu was mostly found in the organelles (78%), and in the heat-stable proteins and peptides (21%) fractions. The organelle fraction appeared to also be the main target compartment of Cu accumulation in Fe-depleted cells. As Fe levels in the medium were shown to influence Cu homeostasis, we found that C. reinhardtii can cope with this additional stress by utilizing different Cu-binding ligands. Indeed, in addition to expected Cu-binding ligands such as glutathione and phytochelatins, 25 proteins were detected that may also play a role in the Cu-detoxification processes in C. reinhardtii. Our results shed new light on the coping mechanisms of C. reinhardtii when exposed to environmental conditions that induce high rates of Cu accumulation.
Subject(s)
Chlamydomonas reinhardtii , Chlamydomonas reinhardtii/metabolism , Copper/metabolism , Iron/metabolism , Isotopes/metabolism , Ligands , ProteomicsABSTRACT
The causative agent of Chagas disease (CD), Trypanosoma cruzi, claims thousands of lives each year. Current diagnostic tools are insufficient to ensure parasitological detection in chronically infected patients has been achieved. A host-derived metabolic signature able to distinguish CD patients from uninfected individuals and assess antiparasitic treatment efficiency is introduced. Serum samples were collected from chronic CD patients, prior to and three years after treatment, and subjected to untargeted metabolomics analysis against demographically matched CD-negative controls. Five metabolites were confirmed by high-resolution tandem mass spectrometry. Several database matches for sex steroids were significantly altered in CD patients. A murine experiment corroborated sex steroid perturbation in T. cruzi-infected mice, particularly in male animals. Proteomics analysis also found increased steroidogenesis in the testes of infected mice. Metabolic alterations identified in this study shed light on the pathogenesis and provide the basis for developing novel assays for the diagnosis and screening of CD patients.
ABSTRACT
Bisphenol A (BPA) metabolism has been investigated using several in vitro models, including human and rat liver microsomes and subcellular (S9) fractions, as well as human-recombinant cytochrome P450 3A4 (CYP3A4) expressed in Supersomes, for a comprehensive look at all possible metabolic pathways. By an untargeted approach using liquid chromatography coupled to a high-resolution quadrupole-time-of-flight mass spectrometer, we were able to detect a large number of known Phase I and Phase II metabolites of BPA, as well as several previously uncharacterized ones. A detailed fragmentation study of BPA and its detected metabolites was crucial to confirm structures. Isotope-labeled BPA analogs were highly useful for the structural elucidation of many metabolites. These results contribute to a better understanding of BPA metabolism, including pathways that may introduce additional toxicity, as well as help with the assessment of BPA exposure in different biological matrices.
Subject(s)
Benzhydryl Compounds/metabolism , Endocrine Disruptors/metabolism , Estrogens/metabolism , Phenols/metabolism , Animals , Chromatography, Liquid , Glucuronides/metabolism , Glutathione/metabolism , Humans , Microsomes, Liver/metabolism , Rats , Sulfates/metabolism , Tandem Mass SpectrometryABSTRACT
Bile acids represent a large class of steroid acids synthesized in the liver and further metabolized by many bacterial and mammalian enzymes. Variations in bile acid levels can be used as a measure of liver function. There still exists, however, a need to study the variation of individual circulating bile acids in the context of hepatotoxity or liver disease. Acetaminophen (APAP), a drug commonly taken to relieve pain and decrease fever, is known to cause acute liver failure at high doses. We have developed a targeted liquid chromatography-tandem mass spectrometry method to monitor the effects of different doses of APAP on the bile acid plasma profile in a rat model. The analysis method was optimized to ensure chromatographic resolution of isomeric species using a mixture of 46 standard bile acids, and 14 isotopically-labeled internal standard (IS) compounds detected in multiple reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer. Four doses of acetaminophen were studied, the highest of which shows signs of hepatotoxicity in rats. This targeted method revealed that high dose APAP has an important effect on bile acid profiles. Changes were seen in several unconjugated bile acids as well as glycine conjugates; however, no obvious changes were apparent for taurine-conjugated species.
ABSTRACT
RATIONALE: Exposure to UV light can induce adverse effects on human health, such as photo-aging, immunosuppression, and cancer. Sunscreens are used to prevent the absorption of UV rays, but certain UV-filtering compounds have been shown to disrupt endocrine systems or act as carcinogens. To assess the effects of the exposure to such compounds, it is important to study the pathways by which they are biotransformed in the body. METHODS: Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC/HRMS/MS) was employed to evaluate the oxidative metabolism and, specifically, the formation of reactive metabolites of six active ingredients commonly used in sunscreen formulations: oxybenzone, avobenzone, homosalate, octisalate, octocrylene, and octinoxate. In vitro incubations were performed with human and rat liver microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate and glutathione. An LC/HRMS/MS method was developed to identify metabolites employing a biphenyl reversed-phase column for separating parent molecules, metabolites, and glutathione (GSH) adducts. RESULTS: Each tested compound resulted in the formation of several metabolites, including at least one GSH adduct. Compounds containing ester groups were hydrolyzed, and some metabolites of the free acid forms were also detected. High-resolution MS/MS data was crucial for the structural elucidation of metabolites and GSH adducts. Fragmentation pathways were proposed for all parent compounds, as well as each described metabolite and adduct. CONCLUSIONS: The results of this study will help better understand the metabolism and detoxification pathways of these xenobiotics.
Subject(s)
Microsomes, Liver/metabolism , Sunscreening Agents/metabolism , Animals , Biotransformation , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Glutathione/metabolism , Humans , Rats , Sunscreening Agents/analysis , Tandem Mass SpectrometryABSTRACT
Light chain amyloidosis (AL) originates from the deposition of immunoglobulin light chains (LCs) as amyloid fibrils in the extracellular space of vital organs. Although non-enzymatic post-translational modifications (PTMs) have been shown to contribute to protein misfolding diseases, little is known about their contributions to LC amyloidogenicity. In this study, we investigated the effects of three oxidative PTMs, carbonylation by hydroxynonenal (HNE), oxidation and nitration, on the structure, thermodynamic stability and self-assembly propensity of a LC variable domain from the λ6 germline, Wil. We initially identified the specific residues that are susceptible to oxidative chemical modifications. HNE-conjugation at specific His residues and nitration of Tyr side chains modulated the conformational conversion driving Wil self-assembly and fibrillar aggregates formation. This study reinforces the notion that not only the thermodynamic stability, but also the chemical and structural properties, should be considered when evaluating the amyloidogenic potential of a LC.
Subject(s)
Amyloid/chemistry , Immunoglobulin Light Chains/chemistry , Protein Processing, Post-Translational , Amyloidosis/etiology , Cell Line , Escherichia coli/genetics , Genetic Vectors , Humans , Immunoglobulin Light Chains/genetics , Male , Molecular Structure , Nitrates/chemistry , Oxidation-Reduction , Oxidative Stress , Protein Aggregates , Protein Carbonylation , Protein StabilityABSTRACT
RATIONALE: An isotopic labeling strategy based on derivatizing amine-containing metabolites has been developed using light ((12) C6 ) and heavy ((13) C6 ) N-benzoyloxysuccinimide reagents for semi-targeted metabolomic applications. METHODS: Differentially labeled samples were combined and analyzed simultaneously by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS) to compare relative amounts of amine-containing metabolites. The selectivity of the reaction was determined with model metabolites and was shown to also be applicable to thiol and phenol moieties. The potential for relative quantitation was evaluated in cell extracts and the method was then applied to quantify metabolic perturbations occurring in human cultured cells under normal vs. oxidative stress conditions. RESULTS: A total of 279 derivatized features were detected in HL60 cell extracts, 77 of which yielded significant concentration changes upon oxidative stress treatment. Based on accurate mass measurements and MS/MS spectral matching with reference standard solutions, 10 metabolites were clearly identified. Derivatized compounds were found to have diagnostic fragment ions from the reagent itself, as well as structurally informative ions useful for metabolite identification. CONCLUSIONS: This simple derivatization reaction can be applied to the relative quantitation of amine-, thiol- and phenol-containing compounds, with improved sensitivity and chromatographic peak shapes due to the increased hydrophobicity of polar metabolites not readily amenable to reversed-phase LC/MS analysis.
Subject(s)
Chromatography, Liquid/methods , Metabolomics/methods , Succinimides/chemistry , Tandem Mass Spectrometry/methods , Carbon Isotopes/analysis , Carbon Isotopes/metabolism , HL-60 Cells , Humans , Isotope LabelingABSTRACT
Rat, mouse and human liver microsomes and S9 fractions were analyzed using an optimized method combining ion exchange fractionation of digested peptides, and ultra-high performance liquid chromatography (UHPLC) coupled to high resolution tandem mass spectrometry (HR-MS/MS). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaíno et al., 2013 [1]) with the dataset identifiers PXD000717, PXD000720, PXD000721, PXD000731, PXD000733 and PXD000734. Data related to the peptides (trypsin digests only) were also uploaded to Peptide Atlas (Farrah et al., 2013 [2]) and are available with the dataset identifiers PASS00407, PASS00409, PASS00411, PASS00412, PASS00413 and PASS00414. The present dataset is associated with a research article published in EuPA Open Proteomics [3].
ABSTRACT
The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.