Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 14kDa. Proteins were positively identified by analysis of the pI (±0.5 pI units), an intact protein molecular weight (±150 14ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 Β, HINT and Α-enolase. Sequence modifications or conflicts were observed for Β-and Γ-actin, ATP Β-synthase and heat shock 90 Β. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters. Copyright © 2001 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35080/1/421_ftp.pd

Resolução lamelar num novo microscópio eletrônico de varredura Lamellar resolution in a new scanning electron microscope

RESUMO: Trabalhando com elétrons de baixa energia (na faixa de 1keV), o novo microscópio eletrônico de varredura dispensa a etapa de metalização e permite a observação direta da estrutura lamelar de polímeros semicristalinos, sem a necessidade de preparação de amostras. São apresentados exemplos da morfologia lamelar do PVDF em função das condições de processamento e da temperatura de cristalização, em filmes contendo as fases a, b e g. Um outro exemplo revela o crescimento inicial da camada transcristalina que se formou ao longo de uma fibra de polietileno de ultra-alto peso molecular embutida em matriz de polietileno de alta densidade.<br>ABSTRACT: Working with low energy electrons (in the range of 1keV), the new scanning electron microscope permits the lamellar (supermolecular) structure of semicrystalline polymers to be observed directly without the need of specimen coating or of any other sample preparation technique. Microscope performance is demonstrated by several examples of high resolution micrographs which show spherulitic, lamellar and fibrilar morphologies developed by the a, b and g phases of PVDF as a function of processing conditions and crystallization temperature. Another example reveals the early stages of transcrystalline layer formation in HDPE reinforced by UHMWPE fibers