Proteins from breast cancer cell lines are characterized using a 2-D liquid separation technique in which protein pI is used as the first-dimension separation parameter. To effect this protein pI separation, chromatofocusing(CF) is employed whereby a pH gradient is generated on-column using a weak anion exchange medium with the intact proteins fractionated and collected every 0.2 pH unit. It is demonstrated that the pI for expressed intact proteins as generated by CF is an important parameter for identification and characterization of the actual protein modifications occurring in the cancer cell. For most proteins, the experimentally determined pI is very close to that predicted by the databases. In other cases, however, where the pI is observed to be shifted from the expected value, it is shown that this shift is often correlated to protein modifications. The modifications that cause such shifts include truncations and deletions often observed in cancer cells or phosphorylations that can shift the pI by several pH units. It is also shown that the effects of phosphorylation on the observed shift can vary depending upon the protein and the amount of phosphorylation. Moreover, large changes in the pI are often observed for proteins with a pI above 7.0 upon phosphorylation, whereas little change is observed for proteins with a pI of approximately 5.0. The expressed protein's pI value thus becomes an important parameter together with the intact MW value, peptide map, and MS/MS results for identification of the presence and type of posttranslational modifications occurring in the cancer cell.

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A multidimensional chromatographic 2-D liquid-phase separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of protein expression between Peninsularinone-treated and untreated HCT-116 human colon adenocarcinoma cells. The method involves fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous reversed-phase HPLC. A 2-D map of the protein content of each cell line based upon pI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or downregulated proteins displayed using ProteoVue and DeltaVue software. Using this method, > 1000 protein bands could be detected in 0.2 pH fractions over a pH range of 4-7. In addition, the liquid eluent from the separation was directed on-line into an electrospray TOF-MS to obtain an accurate molecular weight of the intact proteins. An accurate molecular weight together with the peptide map was used to obtain protein identification using database searching. The method has been shown to have high reproducibility for quantitative differential display analysis of interlysate comparisons, generation of accurate protein identifications, and ease of data interpretation. It has been used herein to identify proteins that change as a function of drug treatment. The relative simplicity of the current procedure and the potential for full automation will make this technique an essential tool in future proteomic studies.

BackgroundEndogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.MethodsFITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis.ResultsPIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.ConclusionCollectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.

Objective Simultaneous analysis of the protein composition of biological fluids is now possible. Such an approach can be used to identify biological markers of disease and to understand the pathophysiology of disorders that have eluded classification, diagnosis, and treatment. The purpose of this study was to analyze the differences in protein composition in amniotic fluid of patients in preterm labor. Study Design Amniotic fluid was obtained by amniocenteses from three groups of women with preterm labor and intact membranes: (1) women without intra-amniotic infection/inflammation (IAI) who delivered at term; (2) women without intra-amniotic IAI who delivered a preterm neonate; and (3) women with IAI. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 (≥2.3 ng/mL). Two-dimensional (2D) chromatography was used for analysis. The first dimension separated proteins by isoelectric point, while the second, by the degree of hydrophobicity. 2D protein maps were generated using different experimental conditions (reducing agents as well as protein concentration). The maps were used to discern subsets of isoelectric point/hydrophobicity containing differentially expressed proteins. Protein identification of differentially expressed fractions was conducted with mass spectrometry. ELISA immunoassays as well as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)--based on-chip antibody capture immunoassay were also used for confirmation of a specific protein that was differentially expressed. Results 1) Amniotic fluid protein composition can be analyzed using a combination of 2D liquid chromatography and mass spectrometry for the identification of proteins differentially expressed in patients in preterm labor; 2) While total insulin-like growth factor-binding protein-1 (IGFBP-1) concentration did not change, IGFBP-1 fragments at about 13.5 kDa were present in patients with intra-amniotic IAI; 3) proteins which were over-expressed in group 1 included Von Ebner gland protein precursor, IL-7 precursor, apolipoprotein A1, tropomyosin sk1 (TPMsk1) fragment, ribosomal protein S6 kinase alpha-3 and alpha-1-microglobulin/bikunin precursor (AMBP); 4) proteins which were over-expressed in group 3 included fibrinopeptide B, transferrin, (MHC) class 1 chain-related A antigen fragment, transcription elongation factor A, sex-determining region Y (SRY) box 5 protein, Down syndrome critical region 2 protein (DSCR2), and human peptide 8 (HP8); and 5) one protein, retinol binding protein, was over-expressed in women who delivered preterm, regardless of the presence of IAI. Conclusions A combination of techniques involving 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in amniotic fluid of patients with preterm labor. Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa ...