Available online 13 September 2022, 104722

GluC/trypsin double digestion of the propeptide followed by MS and MS/MS determined the disulfide connectivity in a SAPA domain.

A theoretical handy work assigned internal and terminal disulfide linked ions to peak signals in MS/MS spectra.

A pattern 1–3 and 2–4 is found for disulfide bonds in the SAPA domain of the human proSP-B precursor.

The disulphide pattern determined by this SAPA domain differs from the established SAPB kringle domain.

The disulfide bonds formed in the SAPA domain of a recombinant version of the NH2-terminal propeptide (SP-BN) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-BN with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys32-Cys49 and Cys40-Cys55, and we propose a disulfide connectivity pattern of 1–3 and 2–4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z ~ 2136 and ~ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides 24AWTTSSLACAQGPE37 and 45QALQCR50 linked by Cys32-Cys49 and 38FWCQSLE44 and 51ALGHCLQE58 linked by Cys40-Cys55 respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1–3/2–4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BN propeptide, the MS analysis of both digests identified the bond Cys100-Cys112, numbered 7–8, which is coincident with the bond position in the kringle motif.

The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.

SAPA domain

Disulfide bonds

SP-B

NH2-terminal propeptide

MALDI-TOF MS

MS/MS

Collision Induced Dissociation

Matrix Assisted Laser Desorption/Ionization Time of Flight

Tandem Mass Spectrometry

Saposin Like Proteins

Surfactant Protein B precursor

NH2-terminal propeptide of proSP-B

© 2022 The Authors. Published by Elsevier B.V.