VERIFICATION 1)Mass spectrometry' s unsurpassed accuracy and speed for measuring mass allows one to quickly test whether a given protein has been faithfully expressed ) The measurement allows for detection of PCR errors mistranslation errors unwanted modifications( e.g., from attachment of beta mercaptoethanol to thiols or oxidation of methionyl residues), and major protein impurities and byproducts(e.g, from degradation) 3 detecting desired modifications such as phosphorylation 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 16 VERIFICATION 1) Mass spectrometry’s unsurpassed accuracy and speed for measuring mass allows one to quickly test whether a given protein has been faithfully expressed. 2) The measurement allows for detection of PCR errors, mistranslation errors , unwanted modifications (e.g., from attachment of betamercaptoethanol to thiols or oxidation of methionyl residues), and major protein impurities and byproducts(e.g., from degradation). 3) detecting desired modifications such as phosphorylation
DOMAIN ELUCIDATION Numerous computer-aided approaches to help define folding domains are available. These include the use of homologous sequence alignment tools secondary structure prediction software, and various threading algorithms. Progress in genomic sequencing has greatly facilitated homology searching, and there are increasingly available more reliable secondary structure prediction programs In cases of sequences that exhibit low homology alignments or poorly predicted secondary structural elements Limited proteolysis combined with Ms 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 17 DOMAIN ELUCIDATION Numerous computer-aided approaches to help define folding domains are available. These include the use of homologous sequence alignment tools, secondary structure prediction software, and various “threading” algorithms. Progress in genomic sequencing has greatly facilitated homology searching, and there are increasingly available more reliable secondary structure prediction programs. In cases of sequences that exhibit low homology alignments or poorly predicted secondary structural elements. Limited proteolysis combined with MS
Figure 2 Domain elucidation by mmss spacircmtry. Top: Hypcthsical three domin protan Felding domins are r-presantol as a diamend, circle, and hexagon. Interdomain linkages are danced as thin black lines limited proteolysis in solution cleanes within the polypeptide link- ages(arrows)rebasing the indiidual folding domains. Bottow: Single protein domain that his unstructured polypeptide segm nts extending beyond the compuct folling domain. Limited pre- teolysis tn ms off these flexibl chins(arroms) leaving a more compact folding domain. In both cases, miss spectrometry allows rapid, acerate definition of the dommin boundaries 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 18
PROTEIN CRYSTAL ANALYSIS Determining the presence of the Expected Protein Components Examining Alteration of Proteins During Crystallization ASsessing Heavy Metal Incorporation 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 19 PROTEIN CRYSTAL ANALYSIS Determining the Presence of the Expected Protein Components Examining Alteration of Proteins During Crystallization Assessing Heavy Metal Incorporation
Crystal A Crystal B TAP Hauter upn ATFIIF Moer配r TFII H+Aiur iobit H时r iTEP hTEIIR Froth crystl RamIn CrysIS NAOmen ddin DNapimtin a回 1 He igure 4 Diret MALDI mass psircmsrk anilysis of potein cry sials ofthe TFIIB-IBP-DNA temary complex(37) allows dtenminalian of their protein composiion. Crstal A cutin d rBP but ma talib wheras crsil b cantina bath tep and telle 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 20