Nanoflow Liquid Chromatography Research Articles

Evaluation of Disposable Trap Column nanoLC-FAIMS-MS/MS for the Proteomic Analysis of FFPE Tissue.

Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.

Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.

Investigation of the blood proteome in response to spinal cord injury in rodent models

Study designExplanatory and mechanistic study.ObjectivesA better understanding of the ‘whole-body’ response following spinal cord injury (SCI) is needed to guide future research aimed at developing novel therapeutic interventions and identifying prognostic indicators for SCI. This study aimed to characterise the blood proteome following contusion or complete SCI compared to a sham injury in rat models.SettingUnited Kingdom.MethodsPooled blood samples from one and seven days after a contusion (serum; n = 5) or from 14 days and 112 days post-complete transection SCI (plasma; n = 8) and their sham-injured counterparts were subjected to independent iTRAQ nanoflow liquid chromatography tandem mass-spectrometry proteomic analyses. Pathway analyses of the proteins that were differentially abundant between SCI and their matched sham injured counterparts were completed to indicate biological pathways that may be changed in response to SCI.ResultsEleven and 42 proteins were differentially abundant (≥±2.0 FC; p ≤ 0.05) between the contusion SCI and sham injured animals at 24 h and seven days post-injury, respectively. Seven and tweleve proteins were differentially abundant between complete and sham injured rats at 14 and 112 days post-injury, respectively. Acute-phase response signalling and Liver X Receptor/Retinoic X Receptor activation were identified as differentially regulated pathways in both models of SCI.ConclusionsWe have utilised longitudinal preclinical SCI models to provide an insight into the blood proteome changes that result following SCI and to highlight a number of biological pathways of interest for future studies.

Sex-specific plasma lipid profiles of ME/CFS patients and their association with pain, fatigue, and cognitive symptoms

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex illness which disproportionally affects females. This illness is associated with immune and metabolic perturbations that may be influenced by lipid metabolism. We therefore hypothesized that plasma lipids from ME/CFS patients will provide a unique biomarker signature of disturbances in immune, inflammation and metabolic processes associated with ME/CFS.MethodsLipidomic analyses were performed on plasma from a cohort of 50 ME/CFS patients and 50 controls (50% males and similar age and ethnicity per group). Analyses were conducted with nano-flow liquid chromatography (nLC) and high-performance liquid chromatography (HPLC) systems coupled with a high mass accuracy ORBITRAP mass spectrometer, allowing detection of plasma lipid concentration ranges over three orders of magnitude. We examined plasma phospholipids (PL), neutral lipids (NL) and bioactive lipids in ME/CFS patients and controls and examined the influence of sex on the relationship between lipids and ME/CFS diagnosis.ResultsAmong females, levels of total phosphatidylethanolamine (PE), omega-6 arachidonic acid-containing PE, and total hexosylceramides (HexCer) were significantly decreased in ME/CFS compared to controls. In males, levels of total HexCer, monounsaturated PE, phosphatidylinositol (PI), and saturated triglycerides (TG) were increased in ME/CFS patients compared to controls. Additionally, omega-6 linoleic acid-derived oxylipins were significantly increased in male ME/CFS patients versus male controls. Principal component analysis (PCA) identified three major components containing mostly PC and a few PE, PI and SM species—all of which were negatively associated with headache and fatigue severity, irrespective of sex. Correlations of oxylipins, ethanolamides and ME/CFS symptom severity showed that lower concentrations of these lipids corresponded with an increase in the severity of headaches, fatigue and cognitive difficulties and that this association was influenced by sex.ConclusionThe observed sex-specific pattern of dysregulated PL, NL, HexCer and oxylipins in ME/CFS patients suggests a possible role of these lipids in promoting immune dysfunction and inflammation which may be among the underlying factors driving the clinical presentation of fatigue, chronic pain, and cognitive difficulties in ill patients. Further evaluation of lipid metabolism pathways is warranted to better understand ME/CFS pathogenesis.

Utilizing liquid chromatography, ion mobility spectrometry, and mass spectrometry to assess INLIGHT™ derivatized N-linked glycans in biological samples.

Glycosylation is a ubiquitous co- and post-translational modification involved in the sorting, folding, and trafficking of proteins in biological systems; in humans, >50% of gene products are glycosylated with the cellular machinery of glycosylation compromising ~2% of the genome. Perturbations in glycosylation have been implicated in a variety of diseases including neurodegenerative diseases and certain types of cancer. However, understanding the relationship between a glycan and its biological role is often difficult due to the numerous glycan isomers that exist. To address this challenge, nanoflow liquid chromatography, ion mobility spectrometry, and mass spectrometry (nLC-IMS-MS) were combined with the Individuality Normalization when Labeling with the Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy to study a series of glycan standards and those enzymatically released from the glycoproteins horseradish peroxidase, fetuin, and pooled human plasma. The combination of IMS and the natural (NAT) and stable-isotope label (SIL) in the INLIGHT™ strategy provided additional confidence for each glycan identification due to the mobility aligned NAT- and SIL-labeled glycans and further capabilities for isomer examinations. Additionally, molecular trend lines based on the IMS and MS dimensions were investigated for the INLIGHT™ derivatized glycans, facilitating rapid identification of putative glycans in complex biological samples.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03570-7.

Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses.

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.

Plasma proteomic profile associated with platelet dysfunction after trauma

BackgroundCoagulopathic bleeding is a major cause of mortality after trauma, and platelet dysfunction contributes to this problem. The causes of platelet dysfunction are relatively unknown, but a great deal can be learned from the plasma environment about the possible pathways involved. ObjectiveDescribe the changes in plasma proteomic profile associated with platelet dysfunction after trauma. MethodsCitrated blood was collected from severely injured trauma patients at the time of their arrival to the Emergency Department. Samples were collected from 110 patients, and a subset of twenty‐four patients was identified by a preserved (n = 12) or severely impaired (n = 12) platelet aggregation response to five different agonists. Untargeted proteomics was performed by nanoflow liquid chromatography tandem mass spectrometry. Protein abundance levels for each patient were normalized to total protein concentration to control for hemodilution by crystalloid fluid infusion prior to blood draw. ResultsPatients with platelet dysfunction were more severely injured but otherwise demographically similar to those with retained platelet function. Of 232 proteins detected, twelve were significantly different between groups. These proteins fall into several broad categories related to platelet function, including microvascular obstruction with platelet activation, immune activation, and protease activation. ConclusionsThis observational study provides a description of the change in proteomic profile associated with platelet dysfunction after trauma and identifies twelve proteins with the most profound changes. The pathways involving these proteins are salient targets for immediate investigation to better understand platelet dysfunction after trauma and identify targets for intervention.

A thermodynamic study of capillary electrochromatographic retention of aromatic hydrocarbons on a lauryl acrylate porous polymer monolithic column with measured phase ratio.

The phase ratio of a chromatographic system is an important measurement that has long been estimated or calculated, but rarely directly measured. This study utilized a nanoflow liquid chromatography instrument to more accurately measure the phase ratio for a lauryl acrylate porous polymer monolith. Direct measurement of the phase ratio, and its dependence on temperature, allows for a better understanding of the thermodynamics of retention of small analytes. This study investigates the retention of an alkyl benzene series, toluene to octylbenzene, via capillary electrochromatography. The phase ratio was determined to be 0.202 at 303 K and 0.213 at 333K. Using the directly measured phase ratio, entropic contributions to retention can also be obtained. Therefore, the Gibbs free energy calculations from these measurements and methods can give insight to modes of retention. The free energy of retention for toluene is -3.97kJ/mol at 303 K and -3.78kJ/mol at 333K. The trends for enthalpy, entropy, and Gibbs free energy of transfer show that retention is enthalpically driven in this capillary electrochromatography (CEC) porous polymer monolith system.

Proteoglycan profiling of human, rat and mouse insulin-secreting cells.

Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.

Trihydroxycholanoyl-taurine in brains of rodents with hepatic encephalopathy.

Hepatic encephalopathy (HE), a neurological disease resulting from liver failure, is difficult to manage and its causes are unclear. Bile acids have been postulated to be involved in the provenance and progression of various diseases including HE. Hence, the characterization of bile acid profiles in the brains of subjects with and without liver failure can provide important clues for the potential treatment of HE. Nanoflow ultra-performance liquid chromatography electrospray ionization ion mobility mass spectrometry (UPLC-ESI-IM-MS) is a highly sensitive method for detection of specific molecules, such as bile acids in brain samples, at biologically relevant concentrations. We used UPLC-ESI-IM-MS to characterize bile acid profiles in brain samples from seven "healthy" control rodents and 22 "diseased" rodents with liver failure (i.e., induced HE). An isomer of trihydroxycholanoyl-taurine was detected in brain tissue samples from both rats and mice with induced HE; however, this isomer was not detected in the brains of healthy rats and mice. Our findings were confirmed by comparing IM arrival times (AT), exact mass measurements (m/z), and mass spectral fragmentation patterns of the experimentally observed suspected species to standards of trihydroxycholanoyl-taurine isomers. Moreover, In Silico Fractionation was employed to provide an additional analytical dimension to verify bile acid identifications.

Performance of nanoflow liquid chromatography using core-shell particles: A comparison study

Due to its unique structure, core-shell material has presented significantly improved chromatographic performance in comparison with conventional totally porous material. This has been well demonstrated in the analytical column format, e.g. 4.6 mm i.d. columns. In the proteomics field, there is always a demand for high resolution microseparation tools. In order to explore core-shell material's potential in proteomics-oriented microseparations, we investigated chromatographic performance of core-shell material in a nanoLC format, as well as its resolving power for protein digests. The results show core-shell nanoLC columns have similar van Deemter curves to the totally porous particle-packed nanoLC columns. For 100 µm i.d. capillary columns, the core-shell material does not have significantly better dynamics. However, both core-shell and totally porous particle-packed nanoLC columns have shown high efficiencies: plate heights of ~11 µm, equivalent to 90000 plates per meter, have been achieved with 5 µm particles. Using a 60 cm long core-shell nanoLC column, 72000 plates were realized in an isocratic separation of neutral compounds. For a 15 cm long nanoLC column, a maximum peak capacity of 220 has been achieved in a 5 hour gradient separation of protein digests, indicating the high resolving power of core-shell nanoLC columns. With a standard HeLa cell lysate as the sample, 2546 proteins were identified by using the core-shell nanoLC column, while 2916 proteins were identified by using the totally porous particle-packed nanoLC column. Comparing the two sets of proteomics data, it was found that 1830 proteins were identified by both columns, while 1086 and 716 proteins were uniquely identified by using totally porous and core-shell particle-packed nanoLC columns, respectively, suggesting their complementarity in nanoLC-MS based proteomics.

Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry.

Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.

Amidation/non-amidation top-down analysis of endogenous neuropeptide Y in brain tissue by nano flow liquid chromatography orbitrap Fourier transform mass spectrometry.

Neuropeptide Y (NPY) is a transmitter molecule in nerve system, and it was an over 4‐kDa large peptide with the C‐terminal end amidation. NPY is biosynthesized through many maturation processes from a large pre‐pro‐peptide with peptide‐cleavages and amidation that is important to study the biosynthesis regulation. Previously, it was reported that cathepsin L participates in the production of NPY and that cathepsin L generates both of amidated and non‐amidated NPYs. However, the non‐amidated NPY (NPY‐COOH) has not been reported in brain tissues until now. In this study, endogenous NPY‐COOH in mouse brain tissue was detected and identified by using nano flow liquid chromatography (nanoLC) orbitrap Fourier transform mass spectrometry (FT‐MS) after the effective purification and separation of NPY‐COOH from NPY‐amide and other peptides using two different gel‐filtration chromatography. Amidated NPY was eluted earlier than non‐amidated NPY‐COOH in the C18 reversed phase nanoLC and the silica‐based gel‐filtration chromatogram with hydrophobic interaction. The amount of endogenous NPY‐COOH was about 0.05% of the matured NPY‐amide amount in adult mouse brain.

Peptidomics of enteroendocrine cells and characterisation of potential effects of a novel preprogastrin derived-peptide on glucose tolerance in lean mice

ObjectivesTo analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides. MethodsHigh resolution nano-flow liquid chromatography mass spectrometry (LC–MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance. ResultsA large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435−462a) had no measurable effect, but a progastrin-derived peptide (Gast p59−79), modestly improved glucose tolerance in lean mice. ConclusionLC–MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59−79, with minor effects on glucose tolerance.

Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro

Alpha-amanitin (α-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography–tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Mycotoxins in cereals and pulses harvested in Latvia by nanoLC-Orbitrap MS

ABSTRACT Twenty-seven mycotoxins in unprocessed cereals (n = 110) and pulses (n = 23) harvested in Latvia were analysed by nanoflow liquid chromatography combined with Orbitrap high-resolution mass spectrometry. One or more mycotoxins were found in 99% of the cereals and 78% of the pulses. Deoxynivalenol, zearalenone and T-2 and HT-2 toxins were prevalent in 9 to 86% of the cereals, mostly below their maximum levels as set by the European regulations. Non-regulated type A and B trichothecenes were prevalent in 5 to 87% of the cereals, at concentrations of 0.27–83 µg kg−1 and 1.7–4,781 µg kg−1, respectively. Quantification of emerging mycotoxins was also provided. Enniatins were detected in 94% of the cereals (3.5–2,073 µg kg−1) and 13% of the pulses (4.4–17 µg kg−1). Alternaria toxins were prevalent in 94% of the cereals at concentrations of 0.72–307 µg kg−1 and in 39% of the pulses at 0.69–10 µg kg−1.

Phenyl-bonded monolithic silica capillary column liquid chromatographic separation and detection of fluorogenic derivatized intact proteins.

Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, has typically been performed by using a conventional reversed-phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl-bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast (Saccharomyces cerevisiae) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the "spiderweb" chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC–MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of “hard-to-find” disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.

Development of a Novel Minimally Invasive Sampling and Analysis Technique for Bioarchaeological Proteomics

Ancient proteomic analysis has been shown to have several advantages over ancient DNA studies, largely due to the ability of many proteins to survive over longer periods of time than their corresponding genes. High-resolution mass spectrometry allows for large scale data to be generated from small amounts of sample. Typically, sampling of bioarchaeological material for proteomics has involved the use of invasive tools such as drills and hammers, which presents an issue when working with museums and other heritage organisations. There exist a few studies in the published literature on the use of specifically manufactured sampling tapes for the minimally invasive analysis of paintings and frescoes, and recently an example of their application to mummified skin. Here we present the development of a novel minimally invasive sample preparation technique for application in the mass spectrometric analysis of bioarchaeological materials. The extraction protocol was developed by applying commercially available, dermatology grade skin sampling strips to modern skin surfaces as a surrogate, and then applied to cranial and bone fragments belonging to a 26th Dynasty Egyptian mummified individual from the coffin of a woman named Mer-Neith-it-es. Extracted proteins were subjected to electrophoretic separation and proteolytic digestion, resulting in peptides that were separated, fragmented and identified using nanoflow liquid chromatography - high resolution tandem mass spectrometry. We have identified a number of ancient brain and intracellular proteins on the surfaces of the various cranial and bone fragments, without causing any significant damage to these valuable remains.