Automated data interpretation based on the concept of “negative signature mass” for mass-mapping disulfide structures of cystinyl proteins

Abstract

An efficient method for data processing and interpretation is needed to support and extend disulfide mass-mapping methodology based on partial reduction and cyanylation-induced cleavage to proteins containing more than four cystines. Here, the concept of "negative signature mass" is introduced as the novel feature of an algorithm designed to identify the disulfide structure of a cystinyl protein given an input of mass spectral data and an amino acid sequence. The "negative signature mass" process is different from the conventional approach in that it does not directly rule-in disulfide linkages, but rather eliminates linkages from a list of all possible theoretical linkages, with the goal of ruling out enough linkages so that only one disulfide structure can be constructed. The operating principles and the effectiveness of the algorithm are described in the context of analyzing ribonuclease A, a 124-residue protein containing eight cysteines in the form of four cystines (disulfides).

Mass spectrometry-based strategies for protein disulfide bond identification

“…It is especially useful for peptides containing sequential cysteines, and no enzyme can cut in between, thereby, hampering the disulfide bond identification. The concept of "negative signature mass algorithm" (NSMA) was introduced: The obtained mass spectrum contained information of all related pro ducts as indirect evidence that was used to rule out enough disulfide linkages so that only one disulfide structure can be constructed (Qi et al 2003, Wu et al 2004). The difficult part of cyanylation is that the fragments after cleavage may not be detected due to either the variation of reaction efficiency at different locations on peptides or the chemical background interference during MALDI analysis (Wu et al 2004).…”

Algorithm-assisted elucidation of disulfide structure: application of the negative signature mass algorithm to mass-mapping the disulfide structure of the 12-cysteine transforming growth factor β type II receptor extracellular domain

“…(These analyses were performed here to illustrate the complexity and status of the sample at various stages of sample processing; neither preliminary analysis by HPLC nor determination of the state of analyte reduction by analysis by MALDI is a prerequisite for subsequent use of the NSMA for processing the mass spectral data from analysis of CNinduced cleavage products. 6 ) The collected fractions were pooled, treated with an OrgHg solid-phase column, desalted by HPLC, cleaved with aqueous ammonia, and then fully reduced as described below. The Wnal resulting mixture of CN-induced cleavage products was analyzed by MALDI-MS to provide input data for the NSMA algorithm to solve the disulWde structure.…”

“…Such diYculty in isolating the partially reduced and cyanylated isoforms of a cystinyl protein makes it diYcult to use our original strategy of direct analysis [2] to deduce the disulWde structure. However, because use of the NSMA [6] does not require separation of individual partially reduced, cyanylated isoforms (nor knowledge of their state of reduction; see footnote 8), the HPLC fractions were pooled (having been collected only to illustrate the complexity of the mixture), Wltered through an OrgHg column, cleaved in aqueous ammonia, and then completely reduced [2]. The resulting complex mixture of CN-induced cleavage fragments was partially separated by microbore HPLC (data not shown) and collected into 12 fractions.…”

“…3 Hence, manual processing of the mass-mapping data for a protein containing more than eight cysteines is impractical. For this reason, we introduced the concept of a "negative signature mass algorithm" (NSMA) 4 to deduce the disulWde structure of a cystinyl protein given an input of its amino acid sequence and mass spectral data from the partial reduction and cyanylation-induced cleavage mass-mapping experiment [6]. The concept of negative signature mass rules out the existence of theoretically possible linkages based on detecting certain structurally diagnostic cyanylation-induced cleavage fragments of the cystinyl protein.…”

“…The concept of the NSMA is based on detection of certain cyanylation-induced cleavage fragments that serve as negative signature masses (NSMs) to rule out certain disulWde linkages from a list of all theoretically possible linkages for the protein under consideration [6]. By general deWnition, a NSM corresponds to a CNinduced cleavage fragment that contains internal free cysteines, i.e., cysteines that lie between the N and C termini of the given CN-induced cleavage fragment.…”