BIOMEDICAL IMPORTANCE

Column Chromatography

  • 藉由column中beads的物理化學性質,將通過的溶液篩出想要的東西

HPLC—High-Pressure Liquid Chromatography

  • 第一代column chromatography的beads很大顆(0.1 mm),flow的效果較差。而總表面積小,限制了添加charged or ligand-like groups
  • 進化的column的beads較細,但阻力變大,因此需要高壓。高壓會把原本由多醣體製成的beads壓碎,後來有silicon beads才克服

Size-Exclusion Chromatography

  • 根據蛋白質的Stokes radii來做篩選
    • Stokes radii為蛋白質的effective volume occupied,與mass及shape有關
  • Size越小,越易先陷入beads的凹洞,因此越往下size越大

Ion-Exchange Chromatography

  • Beads可做成陽離子及陰離子

Hydrophobic Interaction Chromatography

  • Stationary phase用hydrophobic group做coating

Affinity Chromatography

  • 用ligands

Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)

  • SDS-PAGE
  • SDS = sodium dodecyl sulfate,為陰離子清潔劑。每一個SDS結合兩個peptide bonds,使polypeptide展開或變性,因此可將蛋白質解開,之後再跑電泳

Isoelectric Focusing (IEF)

  • 蛋白質因為電性移動到環境pH等於其isoelectric  point (pI)之處
  • IFE-SDS-PAGE:兩個向量(平面),一個根據電荷質量比做電泳,另一個則根據pI做電泳

  • 胰島素
  • 1-fluoro-2,4-dinitrobenzene為Sanger’s reagent,會和α-amino groups反應,等於是把peptide的α端的胺基酸做標記,因而可知是哪一個胺基酸

  • Phenyl isothiocyanate為Edman reagent,也可標記α端的胺基酸。與Sanger’s reagent不同,此反應可逆,也就是試劑可反覆使用(同一試劑依序定序出胺基酸)
  • 很費工夫,因為要先把蛋白切成許多peptides,然後要靠重疊的部份去判斷出整段的順序

  • DNA sequencing比定序胺基酸更有效率

  • Edman technique已被mass spectrometry (MS)取代做為辨認蛋白的工具

  • 可辨視出posttranslational modification

Peptides Can Be Volatilized for Analysis by Electrospray Ionization or Matrix-Assisted Laser Desorption

Tandem Mass Spectrometry

  • 縮寫:MS-MS或MS2
  • 篩檢新生兒代謝性疾病

The Goal of Proteomics Is to Identify the Entire Complement of Proteins Elaborated by a Cell Under Diverse Conditions

Simultaneous Determination of Hundreds of Proteins Is Technically Challenging

  • 第一代proteomics用SDS-PAGE或two-dimensional電泳,再用Edman技術去辨視蛋白

Bioinformatics Assists Identification of Protein Functions