Top Down Characterization of Larger Proteins (45 kDa) by Electron Capture Dissociation Mass Spectrometry

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• 作者：Ying Ge ; Brian G. Lawhorn ; Mariam ElNaggar ; Erick Strauss ; Joo-Heon Park ; Tadhg P. Begley ; and Fred W. McLafferty
• 刊名：Journal of the American Chemical Society
• 出版年：2002
• 出版时间：January 30, 2002
• 年：2002
• 卷：124
• 期：4
• 页码：672 - 678
• 全文大小：164K
• 年卷期：v.124,no.4(January 30, 2002)
• ISSN：1520-5126

文摘

The structural characterization of proteins expressed from the genome is a major problem inproteomics. The solution to this problem requires the separation of the protein of interest from a complexmixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errorsand posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extendedby the greatly increased protein fragmentation from electron capture dissociation (ECD), has been appliedto characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation ofproline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complexmixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but nonecorresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS,ThiG, and ThiF). MS/MS of one ion species (representing ~1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Furtherpurification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneouslyas ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminalMet and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDaphosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme Abiosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this isdue to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECDshowed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, andMS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) andto formation of disulfide bonds (2 × 2 Da) at C₃₂-C₄₉ and C₁₄₃-C₁₄₇, although 10 S-S positions werepossible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins wasrelatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS forthe detailed structural characterization of large proteins.