Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS.

Laser microdissection (LMD) was utilized for the separation of the yolk, follicular wall (granulosa and theca), and surrounding stromal cells of small white follicles (SWF) obtained from reproductively active domestic fowl. Herein, we provide an in situ proteomics-based approach to studying follicular development through the use of LMD and mass spectrometry. This study resulted in a total of 2889 proteins identified from the three specific isolated compartments. White yolk from the smallest avian follicles resulted in the identification of 1984 proteins, while isolated follicular wall and ovarian stroma yielded 2470 and 2456 proteins, respectively. GO annotations highlighted the functional differences between the compartments. Among the three compartments examined, the relative abundance of vitellogenins, steroidogenic enzymes, anti-Mullerian hormone, transcription factors, and proteins involved in retinoic acid receptors/retinoic acid synthesis, transcription factors, and cell surface receptors such as EGFR and their associated signaling pathways reflected known cellular function of the ovary. This study has provided a global proteome for SWF, white yolk, and ovarian stroma of the avian ovary that can be used as a baseline for future studies and verifies that the coupling of LMD with proteomic analysis can be used to evaluate proteins from small, physiologically functional compartments of complex tissue.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Machine learning reveals sex-specific 17ß-estradiol-responsive expression patterns in white perch (Morone americana) plasma proteins.

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17ß-estradiol (E2 ) induction. Semiquantitative nanoLC-MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two-way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E2 -induced plasma samples using the protein expression data. E2 -induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain-containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E2 -responsive proteins in fishes and therefore may be useful indicators of estrogen induction.

C/EBPα regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint.

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4(Cdt2). Cdt2 is the substrate recognition subunit of CRL4(Cdt2) and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4(Cdt2) mediated degradation of p21.

Accurate identification of deamidated peptides in global proteomics using a quadrupole orbitrap mass spectrometer.

Deamidation of asparagine and glutamine residues is a common post-translational modification. Researchers often rely on mass spectrometric based proteomic techniques for the identification of these post-translational sites. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping (13)C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. For proper assignment, it is inherently important to use a mass spectrometer with high mass measurement accuracy and high resolving power. Herein, mouse brain tissue lysate was prepared using filter-aided sample preparation (FASP) method and Stage Tip fractionation followed by analysis on a nanoLC coupled with a quadrupole orbitrap (Q-Exactive) mass spectrometer to accurately identify more than 5400 proteins. Mass spectral data was processed using MASCOT and ProteoIQ for accurate identification of peptides and proteins. MASCOT search values for precursor and MS/MS mass tolerances were investigated, and it was determined that data searched with greater than 5 ppm precursor mass tolerance resulted in the misassignment of deamidated peptides. Peptides that were identified with a mass measurement accuracy of ±5 ppm were correctly assigned.

Comparative proteomic analysis and IgE binding properties of peanut seed and testa (skin).

To investigate the protein composition and potential allergenicity of peanut testae or skins, proteome analysis was conducted using nanoLC-MS/MS sequencing. Initial amino acid analysis suggested differences in protein compositions between the blanched seed (skins removed) and skin. Phenolic compounds hindered analysis of proteins in skins when the conventional extraction method was used; therefore, phenol extraction of proteins was necessary. A total of 123 proteins were identified in blanched seed and skins, and 83 of the proteins were common between the two structures. The skins contained all of the known peanut allergens in addition to 38 proteins not identified in the seed. Multiple defense proteins with antifungal activity were identified in the skins. Western blotting using sera from peanut-allergic patients revealed that proteins extracted from both the blanched seed and skin bound significant levels of IgE. However, when phenolic compounds were present in the skin protein extract, no IgE binding was observed. These findings indicate that peanut skins contain potentially allergenic proteins; however, the presence of phenolic compounds may attenuate this effect.

Effect of post-excitation radius on ion abundance, mass measurement accuracy, and isotopic distributions in Fourier transform ion cyclotron resonance mass spectrometry.

We report an evaluation of a modern Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) instrument to determine the general trend of post-excitation radius on total ion abundance, mass measurement accuracy, and isotopic distributions for internally calibrated mass spectra. The optimum post-excitation radius was determined using total ion abundance, mass measurement accuracy (MMA), and isotope ratios. However, despite the utility of internal calibration for achieving ultimate MMA, the internal calibrant ions were insufficient for compensating for sub-optimum ICR cell conditions. The findings presented herein underscore the importance of determining the optimal post-excitation radius in FT-ICR-MS to achieve high ion abundance (low limits of detection), high MMA, and valid isotopic distributions.

Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization fourier-transform ion-cyclotron-resonance mass spectrometry.

BACKGROUND: One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction. METHODS: TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography-dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study. RESULTS: Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13,761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (< or = 3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H+]11+ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements. CONCLUSIONS: FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique.

Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.

Coordination-driven self-assembly of supramolecular cages: heteroatom-containing and complementary trigonal prisms.

The self-assembly of three nanoscopic prisms of approximate size 1 x 4 nm is reported. Tetrahedral carbon, silicon, and phosphorus were used as structure-defining elements in these coordination-based cages. A carbon-based assembly completes a pair of nanoscopic complementary 3-D structures. The formation of the structures is supported by multinuclear NMR, ESI FT-ICR mass spectrometry, and elemental analysis data.

Mass spectral determination of skeletal/cardiac actin isoform ratios in cardiac muscle.

Skeletal and cardiac muscle contains actin isoforms that vary by two juxtaposed amino acids and two amino acid substitutions (Met299Leu and Ser358Thr). This close sequence homology does not allow cardiac and skeletal actin isoforms to be resolved in traditional SDS-PAGE analysis as the molecular weights (Deltamass = 32 Da) are not significantly different and the pIs are identical (5.2). Although cardiac actin is the predominant form in cardiac muscle, there appears to be a specific skeletal/cardiac actin ratio in a normal heart that may vary in a compromised or diseased heart. In an effort to ascertain the validity of this hypothesis we developed a mass spectrometric technique to measure the ratio of skeletal to cardiac actin. The technique involves purification of muscle actin and subsequent liquid chromatography coupled with electrospray ionization Fourier transform ion cylcotron resonance (LC/FTICR-MS) mass spectrometry. A 7 Tesla FTICR mass spectrometer was utilized to compare skeletal/cardiac actin isoform ratios. Additionally, a new dual electrospray ionization source was employed to determine accurate masses of the alpha-skeletal and alpha-cardiac actins.

Implications of hydrophobicity and free energy of solvation for characterization of nucleic acids by electrospray ionization mass spectrometry.

Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%.