No abstract

We have used a proteomics subcellular spatial razor approach to look at changes in total protein abundance and in protein distribution between the nucleus and cytoplasm following exposure of MCF7 breast cancer cells to estradiol. The dominant response of MCF7 cells to estrogen stimulation involves dynamic changes in protein subcellular spatial distribution rather than changes in total protein abundance. Of the 3604 quantitatively monitored proteins, only about 2% show substantial changes in total abundance (>2-fold), whereas about 20% of the proteins show substantial changes in local abundance and/or redistribution of their subcellular location, with up to 16-fold changes in their local concentration in the nucleus or the cytoplasm. We propose that dynamic redistribution of the subcellular location of multiple proteins in response to stimuli is a fundamental characteristic of cells and suggest that perturbation of cellular spatial control may be an important feature of cancer.

In the last decade or so, polychlorinated biphenyls (PCBs) garnered renewed attention in the scientific community due to new evidence pointing at their continued presence in the environment and workplaces and the potential human risks related to their presence. PCBs move from the environment to humans through different routes; the dominant pathway is the ingestion of contaminated foods (fish, seafood and dairy products), followed by inhalation (both indoor and outdoor air), and, to a lesser extent, dust ingestion and dermal contact. Numerous studies reported the environmental and occupational exposure to these pollutants, deriving from building materials (flame-retardants, plasticizers, paints, caulking compounds, sealants, fluorescent light ballasts, etc.) and electrical equipment. The highest PCBs contaminations were detected in e-waste recycling sites, suggesting the need for the implementation of remediation strategies of such polluted areas to safeguard the health of workers and local populations. Furthermore, a significant correlation between PCB exposure and increased blood PCB concentrations was observed in people working in PCB-contaminated workplaces. Several epidemiological studies suggest that environmental and occupational exposure to high concentrations of PCBs is associated with different health outcomes, such as neuropsychological and neurobehavioral deficits, dementia, immune system dysfunctions, cardiovascular diseases and cancer. In addition, recent studies indicate that PCBs bioaccumulation can reduce fertility, with harmful effects on the reproductive system that can be passed to offspring. In the near future, further studies are needed to assess the real effects of PCBs exposure at low concentrations for prolonged exposure in workplaces and specific indoor environments.

The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the alpha(s1)-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine alpha(s1)-CN A and B variants, which differ by a Pro(16) (B)->Leu(16) (A) substitution. MALDI analysis of the digest allowed the detection of alpha(s1)-CN f8-22 and caprine alpha(s1)-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the alpha(s1)-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms.

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α(s2)-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.

The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of ΔNp63α protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest.