Mass transfer of proteins in chromatographic media: Comparison of pure and crude feed solutions.

Elucidation of intraparticle mass transfer mechanisms in protein chromatography is essential for process design. This study investigates the differences of adsorption and diffusion parameters of basic human fibroblast factor 2 (hFGF2) in a simple (purified) and a complex (clarified homogenate) feed solution on the grafted agarose-based strong cation exchanger Capto S. Microscopic investigations using confocal laser scanning microscopy revealed slower intraparticle diffusion of hFGF2 in the clarified homogenate compared to purified hFGF2. Diffusive adsorption fronts indicated a strong contribution of solid diffusion to the overall mass transfer flux. Protein adsorption methods such as batch uptake and shallow bed as well as breakthrough curve experiments confirmed a 40-fold reduction of the mass transfer flux for hFGF2 in the homogenate compared to pure hFGF2. The slower mass transfer was induced by components of the clarified homogenate. Essentially, the increased dynamic viscosity caused by a higher concentration of dsDNA and membrane lipids in the clarified homogenate contributed to this decrease in mass transfer. Moreover, binding capacity for hFGF2 was much lower in the clarified homogenate and substantially decreased the adsorbed phase driving force for mass transfer.

Tetraspanin 5 (TSPAN5), a Novel Gatekeeper of the Tumor Suppressor DLC1 and Myocardin-Related Transcription Factors (MRTFs), Controls HCC Growth and Senescence.

Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16INK4a/pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.

Inhibition of TRPM7 blocks MRTF/SRF-dependent transcriptional and tumorigenic activity.

Myocardin-related transcription factors A and B (MRTFs) are coactivators of Serum Response Factor (SRF) that mediates the expression of genes involved in cell proliferation, migration and differentiation. There is mounting evidence that MRTFs and SRF represent promising targets for hepatocellular carcinoma (HCC) growth. Since MRTF-A nuclear localization is a prerequisite for its transcriptional activity and oncogenic properties, we searched for pharmacologically active compounds able to redistribute MRTF-A to the cytoplasm. We identified NS8593, a negative gating modulator of the transient receptor potential cation channel TRPM7, as a novel inhibitor of MRTF-A nuclear localization and transcriptional activity. Using a pharmacological approach and targeted genome editing, we investigated the functional contribution of TRPM7, a unique ion channel containing a serine-threonine kinase domain, to MRTF transcriptional and tumorigenic activity. We found that TRPM7 function regulates RhoA activity and subsequently actin polymerization, MRTF-A-Filamin A complex formation and MRTF-A/SRF target gene expression. Mechanistically, TRPM7 signaling relies on TRPM7 channel-mediated Mg2+ influx and phosphorylation of RhoA by TRPM7 kinase. Pharmacological blockade of TRPM7 results in oncogene-induced senescence of hepatocellular carcinoma (HCC) cells in vitro and in vivo in HCC xenografts. Hence, inhibition of the TRPM7/MRTF axis emerges as a promising strategy to curb HCC growth.

Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer.

BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

Nucleoporin Nup155 is part of the p53 network in liver cancer.

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.

RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.

Hepatocyte apoptosis is intrinsically linked to chronic liver disease and hepatocarcinogenesis. Conversely, necroptosis of hepatocytes and other liver cell types and its relevance for liver disease is debated. Using liver parenchymal cell (LPC)-specific TGF-beta-activated kinase 1 (TAK1)-deficient (TAK1LPC-KO) mice, which exhibit spontaneous hepatocellular and biliary damage, hepatitis, and early hepatocarcinogenesis, we have investigated the contribution of apoptosis and necroptosis in hepatocyte and cholangiocyte death and their impact on liver disease progression. Here, we provide in vivo evidence showing that TAK1-deficient cholangiocytes undergo spontaneous necroptosis induced primarily by TNFR1 and dependent on RIPK1 kinase activity, RIPK3, and NEMO. In contrast, TAK1-deficient hepatocytes die by FADD-dependent apoptosis, which is not significantly inhibited by LPC-specific RIPK1 deficiency, inhibition of RIPK1 kinase activity, RIPK3 deficiency or combined LPC-specific deletion of TNFR1, TRAILR, and Fas. Accordingly, normal mouse cholangiocytes can undergo necroptosis, while primary hepatocytes are resistant to it and die exclusively by apoptosis upon treatment with cell death-inducing stimuli in vitro, likely due to the differential expression of RIPK3. Interestingly, the genetic modifications that conferred protection from biliary damage also prevented the spontaneous lethality that was often observed in TAK1LPC-KO mice. In the presence of chronic hepatocyte apoptosis, preventing biliary damage delayed but did not avert hepatocarcinogenesis. On the contrary, inhibition of hepatocyte apoptosis fully prevented liver tumorigenesis even in mice with extensive biliary damage. Altogether, our results suggest that using RIPK1 kinase activity inhibitors could be therapeutically useful for cholestatic liver disease patients.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging.

BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

Computed tomography and histopathological findings after embolization with inherently radiopaque 40µm-microspheres, standard 40µm-microspheres and iodized oil in a porcine liver model.

PURPOSE: The present study compared standard computed tomography (CT) and histopathological findings after endovascular embolization using a prototype of inherently radiopaque 40µm-microspheres with both standard 40µm-microspheres and iodized oil in a porcine liver model. MATERIALS AND METHODS: Twelve pigs were divided into six study groups, of two pigs each. Four pigs were embolized with iodized oil alone and four with radiopaque microspheres; two animals in each group were sacrificed at 2 hours and two at 7 days. Two pigs were embolized with radiopaque microspheres and heparin and sacrificed at 7 days. Two pigs were embolized with standard microspheres and sacrificed at 2 hours. CT was performed before and after segmental embolization and before sacrifice at 7 days. The distribution of embolic agent, inflammatory response and tissue necrosis were assessed histopathologically. RESULTS: Radiopaque microspheres and iodized oil were visible on standard CT 2 hours and 7 days after embolization, showing qualitatively comparable arterial and parenchymal enhancement. Quantitatively, the enhancement was more intense for iodized oil. Standard microspheres, delivered without contrast, were not visible by imaging. Radiopaque and standard microspheres similarly occluded subsegmental and interlobular arteries and, to a lesser extent, sinusoids. Iodized oil resulted in the deepest penetration into sinusoids. Necrosis was always observed after embolization with microspheres, but never after embolization with iodized oil. The inflammatory response was mild to moderate for microspheres and moderate to severe for iodized oil. CONCLUSION: Radiopaque 40µm-microspheres are visible on standard CT with qualitatively similar but quantitatively less intense enhancement compared to iodized oil, and with a tendency towards less of an inflammatory reaction than iodized oil. These microspheres also result in tissue necrosis, which was not observed after embolization with iodized oil. Both radiopaque and standard 40µm-microspheres are found within subsegmental and interlobar arteries, as well as in hepatic sinusoids.

Platelet activation parameters and platelet-leucocyte-conjugate formation in glioblastoma multiforme patients.

Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.

Spatial Tissue Proteomics Quantifies Inter- and Intratumor Heterogeneity in Hepatocellular Carcinoma (HCC).

The interpatient variability of tumor proteomes has been investigated on a large scale but many tumors display also intratumoral heterogeneity regarding morphological and genetic features. It remains largely unknown to what extent the local proteome of tumors intrinsically differs. Here, we used hepatocellular carcinoma as a model system to quantify both inter- and intratumor heterogeneity across human patient specimens with spatial resolution. We defined proteomic features that distinguish neoplastic from the directly adjacent nonneoplastic tissue, such as decreased abundance of NADH dehydrogenase complex I. We then demonstrated the existence of intratumoral variations in protein abundance that re-occur across different patient samples, and affect clinically relevant proteins, even in the absence of obvious morphological differences or genetic alterations. Our work demonstrates the suitability and the benefits of using mass spectrometry-based proteomics to analyze diagnostic tumor specimens with spatial resolution. Data are available via ProteomeXchange with identifier PXD007052.

Proteomic Analysis Reveals GMP Synthetase as p53 Repression Target in Liver Cancer.

Disruption of the tumor-suppressive p53 network is a key event in human malignancies, including primary liver cancer. In response to different types of stress, p53 mediates several antiproliferative cellular outcomes, such as cell cycle arrest, apoptosis, and senescence, by activation or repression of its target genes. Metabolic alterations initiating or being part of the p53 response have become an actively studied research area in the p53 field, with several aspects that still remain to be elucidated. Herein, we identified GMP synthetase (GMPS), a key enzyme of de novo purine biosynthesis, as an important p53 repression target using a large-scale proteomics approach. This p53-mediated repression of GMPS could be validated by immunoblotting in Sk-Hep1, HepG2, and HuH6 cells. Moreover, we found GMPS transcriptionally repressed in a p21-dependent manner and its repression maintained in the context of p53-mediated cellular senescence. More important, direct knockdown of GMPS by RNA interference resulted in reduced cell viability and was sufficient to trigger cellular senescence. Finally, by comparing murine hepatocellular carcinomas, which developed in p53 wild-type (+/+) versus p53 null (-/-) mice, we observed higher GMPS expression in the latter, supporting the in vivo relevance of our findings. We conclude that repression of GMPS by p53 through p21 is a functionally relevant part of the p53-mediated senescence program limiting tumor cell growth in liver cancer.

Cellular apoptosis susceptibility (CAS) is linked to integrin ß1 and required for tumor cell migration and invasion in hepatocellular carcinoma (HCC).

Importins and exportins represent an integral part of the nucleocytoplasmic transport machinery with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show de-regulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. By analyzing ~ 1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi leads to de-regulation of integrins, particularly down-regulation of integrin ß1. Consistent with this finding, CAS knockdown resulted in substantially reduced migration and invasion of HCC cell lines as analyzed by 2D 'scratch' and invasion chamber assays, respectively. Supporting the potential in vivo relevance, high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Our data suggest a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype.

Cellular apoptosis susceptibility (CAS) is overexpressed in thyroid carcinoma and maintains tumor cell growth: A potential link to the BRAFV600E mutation.

Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors.

Prosurvival function of the cellular apoptosis susceptibility/importin-α1 transport cycle is repressed by p53 in liver cancer.

UNLABELLED: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.

Determination of fractional synthesis rates of mouse hepatic proteins via metabolic 13C-labeling, MALDI-TOF MS and analysis of relative isotopologue abundances using average masses.

Proteins of a liver extract taken from a metabolically (13)C-labeled mouse were separated by 2D-PAGE and identified after tryptic digestion by MALDI-TOF MS peptide mass fingerprinting. (13)C-Labeling of proteins was achieved by an infusion of U-(13)C-glucose, which is metabolized to labeled nonessential amino acids. The labeling was analyzed using the relative isotopologue abundances of the measured isotope pattern of tryptic peptides and quantified by their increase in the average molecular mass (DeltaAVM). Fractional synthesis rates (FSR) of proteins were determined from corresponding peptides using measured DeltaAVM values as well as DeltaAVM values deduced from tRNA-precursor amino acid labeling, which in turn was derived from proteins showing high (13)C enrichments. The 8-h FSR values of 43 proteins were determined to range from 0 +/- 0.6 to 95 +/- 1%/8 h, with typical errors given as SEM values, which depend on the number of peptides of a specific protein usable for calculation. The method demonstrates that FSR values as an indicator for protein turnover in the liver proteome can be estimated within narrow error margins, providing baseline values from which treatment-dependent deviations could be detected with high statistical certainty.

Three-beam interference is a sensitive measure of the efficacy of macromolecular refinement techniques.

Triplet phases recorded from insulin crystals were used to measure the improvement of phases during model refinement and to quantify the contribution made by each step in the refinement. Conventional amplitude data were recorded to 1.5 A resolution from rhombohedral pig insulin crystals using 1.54 A Cu Kalpha radiation. An initial atomic model and starting phases were obtained from a published structure and the atomic model was refined against the amplitude data using CNS. The refined phases were compared with 800 triplet phases that were measured from similar crystals using a three-beam interference technique and 1.1 A wavelength synchrotron radiation. The solvent region was improved further using a novel density-modification procedure. Calculated triplet phases were obtained from the model after each step in the refinement and were compared with the recorded triplet phases. The average difference between the recorded triplet phases and the calculated triplet phases was used as an unbiased measure of the correctness of the model at each stage in the refinement. The average individual phase error was estimated from discrepancies from triplet phases after each refinement step. Conventional atomic refinement of an approximate starting model reduced the average individual phase error from 21.6 to 14.7 degrees. Improvement of the solvent region, including the difference-map flattening procedure, reduced the individual phase error by a further 2.6 degrees. Modeling the discrete disorder of four amino acids accounted for an additional 0.5 degrees improvement and the final individual phase error was 11.6 degrees.

Protein abundance quantification in embryonic stem cells using incomplete metabolic labelling with 15N amino acids, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and analysis of relative isotopologue abundances of peptides.

An isotope dilution method for protein quantification is presented in the context of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and mass fingerprinting experiments, revealing an unappreciated high reproducibility and accuracy of relative peak intensity measurements. Labelled proteins were generated by growing cells in a medium containing (15)N-enriched amino acids, and were mixed with proteins of natural isotopic composition from control cells in ratios of approximately 0:1, 1:7, 1:2, 2:1, 7:1, and 1:0 (labelled/unlabelled). Mixtures were separated by two-dimensional gel electrophoresis and analysed by MALDI-TOFMS using typical experimental conditions. A linear relationship is demonstrated between the relative isotopologue abundances (RIA values) for particular peaks in the isotopic distribution of tryptic peptide fragments of the proteins, and the mole fractions of labelled proteins in the mixture. Analysis of RIA values (ARIA quantification) for peptides of six typical silver-stained protein spots for the various mixtures could reproduce the experimentally contrived ratios with approximate errors between 4% (2:1 mixture) and about 18% (1:7 mixture). A consideration of error and its propagation is discussed. ARIA does not require complete separation of the isotope patterns of labelled and unlabelled peptides, and is therefore advantageous in combination with all kinds of labelling experiments in biological systems, because it is compatible with minimal metabolic incorporation of labelling reagent. Simulations indicate that the minimum required (15)N enrichment of the total amino acid pool sufficient for ARIA is less than 4%. In an accompanying paper in this issue, we apply ARIA to proteins differentially labelled with isotope-coded alkylation reagents.

Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-acrylamide alkylation tag system.

The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) to calculate the mixing ratios of composites consisting of stable isotope labelled and isotopically natural (unlabelled) proteins, as described in an accompanying paper in this issue. Recently, Sechi (Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698) introduced the use of differential quantitative mass analysis by MALDI-TOFMS using mixtures of standard proteins alkylated prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with either acrylamide (AA) or deuterium-labelled [2,3,3'-D(3)]-acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels ((125)I and (131)I) to quantitatively establish that non-comigration in 2D-PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver-stained 2D-PAGE protein spots from 2D gels loaded with 150 microg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.

The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) to calculate the mixing ratios of composites consisting of stable isotope labelled and isotopically natural (unlabelled) proteins, as described in an accompanying paper in this issue. Recently, Sechi (Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698) introduced the use of differential quantitative mass analysis by MALDI-TOFMS using mixtures of standard proteins alkylated prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with either acrylamide (AA) or deuterium-labelled [2,3,3'-D3 ]-acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels (125 I and 131 I) to quantitatively establish that non-comigration in 2D-PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver-stained 2D-PAGE protein spots from 2D gels loaded with 150 µg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.