Complex Cell Lysates Research Articles

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

Ultrasensitive peptide-based electrochemical detection of protein kinase activity amplified by RAFT polymerization

Since the oversecretion of protein kinases is indicative of multiple human diseases, the screening of their activities is quite important to clinical diagnosis and targeted therapy. In this work, an ultrasensitive peptide-based electrochemical biosensor was presented for the detection of protein kinase activity by using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique as a signal amplification strategy. First, the substrate peptides were tethered to a gold electrode surface via the thiol terminals. After the phosphorylation of substrate peptides by protein kinases, the carboxyl group-containing dithiobenzoates were labeled to the phosphorylated sites via the robust phosphate-Zr4+-carboxylate linkages. Finally, the RAFT polymerization was initiated using ferrocenylmethyl methacrylates (FcMMAs) and dithiobenzoates as the monomers and the RAFT agents, respectively. The grafting of ferrocenyl polymer chains efficiently recruits a great number of electro-active Fc probes to each phosphorylated site, leading to a drastic amplification of the electrochemical signal. With PKA (protein kinase A) as the target, the detection limit of the peptide-based biosensor can be as low as 1.05 mU mL−1. Moreover, it can selectively differentiate the target from other interferents and is applicable for the screening of potential inhibitors as well as the detection of protein kinase activity in complex cell lysates. Therefore, the peptide-based biosensor shows great promise as a universal tool for protein kinase activity detection and inhibitor screening.

Quantifying Ubiquitination Signaling with a Chemical Proteomics Strategy

Stoichiometric analysis of posttranslational modifications is an emerging strategy for absolute quantification of the modification's fractional abundance. In this study, we present a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination, named Isotope‐BAlanced Quantification of Ubiquitination (IBAQ‐Ub). The strategy utilizes a new amine‐reactive chemical tag (AcGG‐NHS) for highly efficient labeling of amine residues. The new chemical tag is structurally homologue to the GG remnant of ubiquitin on modified lysine following trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and the secondary stable isotopic labeling. Quantitative analysis of labeled peptides with HPLC and high resolution mass spectrometry allows unbiased measurements of absolute site‐specific ubiquitination stoichiometry. Using recombinant protein standards and isotopic serial dilution, we showed that the strategy is highly robust, sensitive and accurate. We further designed a SILAC‐based experiment to demonstrate the efficient labeling of lysine with the new AcGG tag in complex cell lysates. Application of the IBAQ‐Ub workflow for the analysis of enriched histone fractions allowed us to measure the stoichiometry of histone H2B C‐terminal ubiquitination. Comparative analysis of stoichiometric dynamics upon proteasome inhibitor treatments showed a dramatic decrease of histone H2B ubiquitination levels and a significant increase of polyubiquitination K48 linkage. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways in cells and diseases.Support or Funding InformationUniversity of Minnesota start‐up fund and National Science Foundation (CHE‐1753154)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

2-Phenylquinoline-Sugar Hybrids as Photoswitchable α-Glucosidase Inhibitors.

Purpose-designed 2-phenylquinoline (PQ)-sugar hybrids 1 and 2 were synthesized and evaluated for their photodegradation activities against an α-glucosidase target. The results indicated that PQ-mannose hybrid 2 selectively and effectively photodegraded α-glucosidase and significantly inhibited its enzymatic activity upon irradiation with long-wavelength UV light in the absence of any additives under neutral and aqueous conditions. Furthermore, 2 selectively and effectively inhibited α-glucosidase activity only with photo-irradiation even in complex cell lysate.

A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria.

The mycobacterial outer membrane, or mycomembrane, is essential for the viability and virulence of Mycobacterium tuberculosis and related pathogens. The mycomembrane is a dynamic structure, whose chemical composition and biophysical properties can change during stress to give an advantage to the bacterium. However, the mechanisms that govern mycomembrane remodeling and their significance to mycobacterial pathogenesis are still not well characterized. Recent studies have shown that trehalose dimycolate (TDM), a major glycolipid of the mycomembrane, is broken down by the mycobacteria-specific enzyme TDM hydrolase (Tdmh) in response to nutrient deprivation, a process which appears to modulate the mycomembrane to increase nutrient acquisition, but at the expense of stress tolerance. Tdmh activity thus balances the growth of M. tuberculosis during infection in a manner that is contingent upon host immunity. Current methods to probe Tdmh activity are limited, impeding the development of inhibitors and the investigation of the role of Tdmh in bacterial growth and persistence. Here, we describe the synthesis and evaluation of FRET-TDM, which is a fluorescence-quenched analogue of TDM that is designed to fluoresce upon hydrolysis by Tdmh and potentially other trehalose ester-degrading hydrolases involved in mycomembrane remodeling. We found that FRET-TDM was efficiently activated in vitro by recombinant Tdmh, generating a 100-fold increase in fluorescence. FRET-TDM was also efficiently activated in the presence of whole cells of Mycobacterium smegmatis and M. tuberculosis, but the observed signal was predominantly Tdmh-independent, suggesting that physiological levels of Tdmh are low and that other mycobacterial enzymes also hydrolyze the probe. The latter notion was confirmed by employing a native protein gel-based fluorescence assay to profile FRET-TDM-activating enzymes from M. smegmatis lysates. On the other hand, FRET-TDM was capable of detecting the activity of Tdmh in cells when it was overexpressed. Together, our data demonstrate that FRET-TDM is a convenient and sensitive in vitro probe of Tdmh activity, which will be beneficial for Tdmh enzymatic characterization and inhibitor screening. In more complex samples, for example, live cells or cell lysates, FRET-TDM can serve as a tool to probe Tdmh activity at elevated enzyme levels, and it may facilitate the identification and characterization of related hydrolases that are involved in mycomembrane remodeling. Our study also provides insights as to how the structure of FRET-TDM or related fluorogenic probes can be optimized to achieve improved specificity and sensitivity for detecting mycobacteria.

Chemically reprogramming the phospho-transfer reaction to crosslink protein kinases to their substrates.

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

Toward multimarker and functional assays from crude cell lysates: controlling spacing and signal amplification in DNA-CT–based bioelectrochemical devices

Abstract DNA-based electrochemical biosensors that rely on charge transport through DNA (DNA-CT) to detect biomarkers of interest have shown great promise in proof-of-principle studies because of their specificity, sensitivity, and low cost. However, this approach has not translated successfully into real clinical applications. One significant barrier has been the need to measure biomarkers in unpurified, complex cell and tissue lysates, which is difficult with DNA-modified electrodes because of crowding and nonspecific adhesion of biomolecules to the surface. Here, we discuss recent achievements in the control of DNA monolayer formation and the amplification of DNA CT signals that help to facilitate the detection of meaningful biomarkers from unprocessed, clinically relevant lysate samples.

Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity

Phosphorylation of proteins catalyzed by protein kinases (PKs) is essential to many biological processes; the sensitive detection of PK activity and the screening of PK inhibitors are thus integral to disease diagnosis and drug discovery. Herein, a highly sensitive biosensor has been fabricated for the electrochemical detection of PK activity by exploiting the electrochemically controlled reversible addition-fragmentation chain transfer (eRAFT) polymerization as a novel amplification strategy. The fabrication of the eRAFT-polymerization-based electrochemical biosensor involves (1) the immobilization of substrate peptides onto a gold electrode by way of gold-sulfur self-assembly, (2) the site-specific phosphorylation of substrate peptides by PKs, (3) the anchoring of carboxyl-group-containing chain transfer agents (CTAs) to the phosphorylated sites, and (4) the eRAFT polymerization under a potentiostatic condition, using ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through the eRAFT polymerization, long polymer chains containing numerous electroactive Fc tags can be de novo grafted from each phosphorylated site, resulting in significant amplification of the electrochemical detection signal. The as-fabricated biosensor is highly selective and features a very low detection limit of 1.02 mU mL-1, in the presence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent PK (PKA) as the model target. Results also demonstrate that it can be applied to the screening of PK inhibitors and the detection of PK activity in complex serum samples and cell lysates. Moreover, it holds the merits of easy fabrication, high efficiency, and low cost, which make it a promising tool for the detection of PK activity and the screening of potential PK inhibitors.

Plasmonic metal nanoparticles as efficient mass tags for ion signal amplification and ultrasensitive detection of protein markers

The development of sensitive and specific analytical methods is critical for the discovery of molecular biomarkers, which assists disease diagnosis and understanding biological processes. Herein, a highly sensitive method is developed using antibody-conjugated plasmonic metal nanoparticles for the detection of targeted biomarkers down to low attomole level via coupling of immunoassay techniques with laser ionization mass spectrometry (LI-MS). The conjugated antibodies target specific antigens, while the metal nanoparticles act as mass tags and ion reservoirs for the signal amplification. With the characteristic localized surface plasmon resonance (LSPR) properties, gold (AuNPs) and silver nanoparticles (AgNPs) undergo explosive ionization upon laser irradiation to generate abundant characteristic mass reporter ions for strong MS signal amplification. With the antibody-conjugated NPs, detection of trace proteins in various biological samples with complex matrix environment, including urine, cell lysates, and animal tissues was demonstrated.

Carboxymethylated polyethylenimine modified magnetic nanoparticles specifically for purification of His-tagged protein.

Employing immobilized metal-ion affinity chromatography and magnetic separation could ideally provide a useful analytical strategy for purifying His-tagged protein. In the current study, a facile route was designed to prepare CMPEI-Ni2+ @SiO2 @Fe3 O4 (CMPEI=carboxymethylated polyethyleneimine) magnetic nanoparticles composed of a strong magnetic core of Fe3 O4 and a Ni2+ -immobilized carboxymethylated polyethyleneimine coated outside shell, which was formed by electrostatic interactions between polyanionic electrolyte of carboxymethylated polyethyleneimine and positively charged surface of 3-(trimethoxysilyl)propylamin modified SiO2 @Fe3 O4 . The resulting CMPEI-Ni2+ @SiO2 @Fe3 O4 composite nanoparticles displayed well-uniform structure and high magnetic responsiveness. Hexa His-tagged peptides and purified His-tagged recombinant retinoid X receptor alpha were chosen as the model samples to evaluate the adsorption, capacity, and reusability of the composite nanoparticles. The results demonstrated the CMPEI-Ni2+ @SiO2 @Fe3 O4 nanoparticles possessed rapid adsorption, large capacity, and good recyclability. The obtained nanoparticles were further used to purify His-tagged protein in practical environment. It was found that the nanoparticles could selectively capture His-tagged recombinant retinoid X receptor protein from complex cell lysate. Owing to its easy synthesis, large binding capacity, and good reusability, the prepared CMPEI-Ni2+ @SiO2 @Fe3 O4 magnetic nanoparticles have great potential for application in biotechnological fields.

A Quantitative Chemical Proteomics Approach for Site‐specific Stoichiometry Analysis of Ubiquitination

Stoichiometric analysis of post-translational modifications is an emerging strategy for absolute quantification of the fractional abundance of the modification. Herein, a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination is reported, named isotopically balanced quantification of ubiquitination (IBAQ-Ub). The strategy utilizes a new amine-reactive chemical tag (AcGG-NHS) that is structurally homologous to the GG remnant of ubiquitin on modified lysine after trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and secondary stable isotopic labeling. The strategy is highly robust, sensitive, and accurate with a wide dynamic range using either protein standards or complex cell lysates. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways.

A Quantitative Chemical Proteomics Approach for Site‐specific Stoichiometry Analysis of Ubiquitination

AbstractStoichiometric analysis of post‐translational modifications is an emerging strategy for absolute quantification of the fractional abundance of the modification. Herein, a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination is reported, named isotopically balanced quantification of ubiquitination (IBAQ‐Ub). The strategy utilizes a new amine‐reactive chemical tag (AcGG‐NHS) that is structurally homologous to the GG remnant of ubiquitin on modified lysine after trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and secondary stable isotopic labeling. The strategy is highly robust, sensitive, and accurate with a wide dynamic range using either protein standards or complex cell lysates. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways.

Membrane‐Targetable Probes for Hg2+ Detection in Live Cells and Paper‐Based Devices

AbstractTwo novel fluorogenic probes (HGMem‐2 and −3) for detecting Hg2+ were designed, synthesized and characterized. Probes based on a commercially available fluorophore (fluorescein) with high brightness were equipped with/without a photolabile group (2‐nitrobenzyl group), a membrane‐anchoring unit (cholesterol) and a Hg2+‐reactive moiety. Taking advantage of the hydrophobicity of the cholesterol group in the probe, a paper‐based device spotted with HGMem‐2 was developed as a Hg2+ paper indicators, and showed feasibility in complex cell lysate. Only upon UV photolysis, HGMem‐3 displayed more than 1900‐fold fluorescence enhancement with the addition of Hg2+. Furthermore, they exhibited pronounced Hg2+‐selective fluorogenic signalling behavior over other 45 analytes. Importantly, HGMem‐3 was utilized to specifically visualize Hg2+ near cell membrane in live HepG2 cells with high spatiotemporal resolution, avoiding the influence of reactive species outside cells successfully. Hence, our newly developed membrane‐anchored Hg2+ sensors provide useful tools for versatile applications in biological and environmental research.

Development and validation of an improved diced electrophoresis gel assay cutter-plate system for enzymomics studies

Diced electrophoresis gel (DEG) assay is a methodology to identify enzymes with a specified activity in complex cell or tissue lysates by means of two-dimensional separation using isoelectric focusing and native PAGE, followed by dicing of the gel into small pieces that are assayed separately, and digestion and peptide fingerprinting to identify the protein(s) of interest in positive wells. The existing hand-made system has some disadvantages, and here we describe the development and validation of an improved cutter-plate system that enables simple, reliable and reproducible DEG assay in a 384-well plate-based format with signal readout using fluorometric or LC-MS-based reaction monitoring. To illustrate the usefulness of this system, we describe its application to profile esterase activities in ovarian adenocarcinoma SKOV3 cell lysate and mouse liver lysate that activate a fluorogenic substrate, fluorescein dibutyrate (FDBu), as well as esterase activities in mouse liver lysate that activate S-bromobenzylglutathione dicyclopentyl ester (BBGDC), a prodrug of anti-tumor agent S-bromobenzylglutathione. The activity spot patterns detected for FDBu and BBGDC were completely different, indicating that different metabolic systems are involved in hydrolysis of these substrates. The major detected spot in each case was identified. The developed system provides a highly reproducible general assay platform that should be useful for characterizing novel protein functions in complex bio-samples, as well as enzymomics studies.

Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics.

Recent progress in top-down proteomics has driven the demand for chromatographic methods compatible with mass spectrometry (MS) that can separate intact proteins. Hydrophilic interaction liquid chromatography (HILIC) has recently shown good potential for the characterization of glycoforms of intact proteins. In the present study, we demonstrate that HILIC can separate a wide range of proteins exhibiting orthogonal selectivity with respect to reversed-phase LC (RPLC). However, the application of HILIC to the analysis of low abundance proteins (e.g., in proteomics analysis) is hampered by low volume loadability, hindering down-scaling of the method to column diameters below 2.1 mm. Moreover, HILIC-MS sensitivity is decreased due to ion suppression from the trifluoroacetic acid (TFA) often used as the ion-pair agent to improve the selectivity and efficiency in the analysis of glycoproteins. Here, we introduce a capillary-based HILIC-MS method that overcomes these problems. Our method uses RPLC trap-columns to load and inject the sample, circumventing issues of protein solubility and volume loadability in capillary columns (200 μm ID). The low flow rates and use of a dopant gas in the electrospray interface improve protein-ionization efficiencies and reduce suppression by TFA. Overall, this allows the separation and detection of small protein quantities (down to 5 ng injected on column) as indicated by the analysis of a mixture of model proteins. The potential of the new capillary HILIC-MS is demonstrated by the analysis of a complex cell lysate.

Immunoprecipitation.

This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody bound to a solid matrix. Purified antigen(s) can be eluted by various methods, and the resultant protein target can be analyzed and/or identified by numerous assays, including the enzyme-linked immunosorbent assay (ELISA), western blotting, or mass spectrometry.

Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA Biosensing via Target-Triggered Ru(III) Release and Redox Recycling.

A new label-free and enzyme-free ratiometric homogeneous electrochemical microRNA biosensing platform was constructed via target-triggered Ru(III) release and redox recycling. To design the effective ratiometric dual-signal strategy, [Ru(NH3)6]3+ (Ru(III)), as one of the electroactive probes, was ingeniously entrapped in the pores of the positively charged mesoporous silica nanoparticle (PMSN), and another electroactive probe, [Fe(CN)6]3- (Fe(III)), was selected to facilitate Ru(III) redox recycling due to its distinctly separated reduction potential and different redox properties. Owing to the liberation of the formed RNA-ssDNA complex from PMSN, the target miRNA triggered the Ru(III) release and was quickly electroreduced to Ru(II), and then, the in-site-generated Ru(II) could be chemically oxidized back to Ru(III) by Fe(III). Thus, with the release of Ru(III) and the consumption of Fe(III), a significant enhancement for the ratio of electroreduction current [Ru(NH3)6]3+ over [Fe(CN)6]3- (IRu(III)/IFe(III)) value was observed, which was dependent on the concentration of the target miRNA. Consequently, a simple, accurate, and ultrasensitive method for the miRNA assay was readily realized. Furthermore, the limit of detection (LOD) of our method was down to 33 aM (S/N = 3), comparable or even superior to other approaches reported in literature. More importantly, it also exhibited excellent analytical performance in the complex biological matrix cell lysates. Therefore, this homogeneous biosensing strategy not only provides an ingenious idea for realizing simple, rapid, reliable, and ultrasensitive bioassays but also has a great potential to be adopted as a powerful tool for precision medicine.

A label-free and highly sensitive strategy for uracil-DNA glycosylase activity detection based on stem-loop primer-mediated exponential amplification (SPEA)

Uracil-DNA glycosylase (UDG) plays essential roles in base excision repair (BER) pathway by eliminating uracil from DNA to sustain the genome integrity. Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications. We develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification (SPEA). In the presence of active UDG, the uracil base in helper hairpin probe (HP) can be excised to generate an abasic site (AP site), which can be cleaved by endonuclease IV (Endo IV) with a blocked primer released. This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA, which can initiate a loop-mediated isothermal amplification (LAMP) reaction. This reaction generates a large number of long double-strand DNA replicates, which can be stained by SYBR Green (SG) I to deliver enhanced fluorescence for quantitative detection of UDG activity. A linear range from 0.001 U/mL to 1 U/mL and a detection limit down to 0.00068 U/mL are achieved. This strategy has also been demonstrated for UDG assay in complex cell lysates, implying its great potential for UDG based clinical diagnostics and therapeutics.

Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer.

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. In addition to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss-triggered ET(ca/hc)D) and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high-resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlight that a charge-state-dependent fragmentation regime and a multiple algorithm search strategy are likely to be of benefit for confident large-scale phosphosite localization.

A sensitive method for the separation and quantification of low-level adenine nucleotides using porous graphitic carbon-based liquid chromatography and tandem mass spectrometry.

A liquid chromatography coupled to heated electrospray ionization/tandem mass spectrometry (LC-HESI-MS/MS) method was developed for the simultaneous quantitative analysis of low nanomolar level adenine nucleotides AMP, ADP, ATP, cyclic AMP (cAMP), and the nucleoside adenosine. For analyte retention and separation, reverse phase chromatography using porous graphitic carbon (PGC) was employed as it provided full resolution. The erratic chromatographic behaviour characteristic of PGC, including deterioration of analyte resolution and increased peak tailing (leading to decreased sensitivity), was mitigated by incorporating acidic equilibration within runs using a quaternary gradient. Analyte resolution and chromatographic sensitivity were still lost after a period of column inactivity; hence a pre-conditioning protocol was implemented between batches to regenerate the column. These column regeneration measures also allowed elution of AMP, ADP and ATP in the sequence of mono- to tri- nucleotides, differing from conventional reverse phase elution where analytes elute with decreasing polarity. This nucleotide elution sequence has the advantage of overcoming potential mis-annotation and inaccurate quantification of smaller nucleotides caused by in-source fragmentation of ATP. The method was validated in granulosa cell conditioned media, with the LLOQs ranging between 10-50nM for most analytes. To verify the method using biological samples, nucleotide secretion was measured in granulosa cell conditioned media under various treatments known to alter their levels. Moreover, the method was applied to cumulus-oocyte complex cell lysates to examine its linearity in a complex matrix.