Extending Top-Down Mass Spectrometry to Proteins with Masses Greater Than 200 Kilodaltons
Tóm tắt
For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the ∼500-residue, ∼50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).
Từ khóa
Tài liệu tham khảo
Unless stated otherwise proteins were from Sigma-Aldrich (St. Louis MO) and desalted with a Michrom BioResources (Auburn CA) protein trap. Protein solutions were ∼5 μM in 50:45:5 MeCN:H 2 O:AcOH; ∼1 s of ESI is necessary to fill the FTMS cell for measurement of each scan with ∼7 × 10 –13 mol consumed for a 50-scan spectrum. Apparatus (Finnigan 6T FTMS) and experimental details were described recently ( 17 ). There temperatures of the inlet capillary were measured at its flared entrance end just outside the heating current connection; here temperatures were measured in the stainless-steel body with comparative values of 56°/58° 62°/90° 76°/150° and 100°/190°C. Also as justified there ( 17 ) the predicted sequence is used to assign the observed masses to the N- and C-terminal products (b and y ions respectively) and to internal ions (i b and i y ) from secondary b y dissociation. Excluding minor ions from H 2 O/NH 3 loss this approach allowed >95% assignment of observed mass values although obviously the b y are more reliable than the i b and i y assignments. Singly charged fragment ions of low but similar relative intensities (∼1%; up to 10% at T cap = 190°C V pre = 100 V post = 13) appear at nearly every mass from 600 to 1500 daltons even with ESI solution additives. The FTMS resolving power allows THRASH ( 20 ) to recognize and subtract these ions ( 17 ).
A similar top-down approach successfully identified five deamidation (–NH 2 → –OH +1 dalton) sites in ribonuclease A (13.7 kD) ( 28 ).
We thank S. Ealick and L. Quadri for purified PurL and Mas. We also thank them and B. Baird T. Begley H. Zhai and G. Infusini for helpful discussions. This work was supported by the Institute of General Medical Sciences (NIH grant GM16609 to F.W.M.) and the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung and Tiroler Wissenschaftsfonds (grant T229 and UNI-0404/158 to K.B.).