BIOMEDICAL IMPORTANCE

PROTEINS & PEPTIDES MUST BE PURIFIEDPRIOR TO ANALYSIS

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

SANGER WAS THE FIRST TO DETERMINE THE SEQUENCE OF A POLYPEPTIDE

• 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

THE EDMAN REACTION ENABLES PEPTIDES & PROTEINS TO BE SEQUENCED

• 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

MOLECULAR BIOLOGY REVOLUTIONIZED THE DETERMINATION OF PRIMARY STRUCTURE

• DNA sequencing比定序胺基酸更有效率

GENOMICS ENABLES PROTEINS TO BE IDENTIFIED FROM SMALL AMOUNTS OF SEQUENCE DATA

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

MASS SPECTROMETRY CAN DETECT COVALENT MODIFICATIONS

• 可辨視出posttranslational modification

MASS SPECTROMETERS COME IN VARIOUS CONFIGURATIONS

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

PROTEOMICS & THE PROTEOME

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