Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Abstract

Neurotrauma, as in the case of traumatic brain injury, promotes protease over-activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post-injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post-injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post-injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain-cleaved MBP into CSF was verified following brain injury. We ascertained that the exon-6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post-TBI cleavage event within exon-2 and for the dimerization of the post-TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.

Figures

Fig. 1

Rat MBP isoforms and marker peptides. Indicated are the sequences for each isoform (#1–8), the calpain cleavage site (vertical red line) with associated proteolytic fragments (A–F), and the select marker peptides of myelin basic protein (underlined and colored). The molecular mass and associated variable transcription regions of the eight known isoforms and six predicted calpain fragments of MBP are tabulated. Two peptides used for IDA quantification analysis, HGFLPR (magenta) and KNIVTPR (red), are shown in bold. Five exon specific peptides used to differentiate the MBP isoforms are listed. The boxed sequence indicates the epitope for the MBP antibody used in this and earlier studies (Chemicon MAB381).

Fig. 2

Immunoblot analysis of proteolyzed MBP following TBI. (a) Naïve and TBI rat cortex samples resolved by gel electrophoresis were probed with an antibody (MAB381; Chemicon) selective for the exon-6 coded region of MBP. The Western blot illustrates the dramatic reduction of the 21.6 and 18.6 kDa isoforms of MBP following TBI, with a concomitant rise in MBP fragments at and below an Ma of 10. This data correlates with that ofAkiyama et al. (2002) and Liu et al. (2006a), where the same antibody was used to characterize MBP in rat cortex. (b) CSF and TBI samples were resolved along side one another by gel electrophoresis and were probed with the MBP antibody. The MBP fragments observed in the TBI tissue were not detected in TBI CSF. The only band (Ma of 12) in the CSF samples is adjacent to a non-specifically detected band of hemoglobin (Hb) observed in TBI tissue (blood contamination).

Fig. 3

Data for peptide quantification by tandem mass spectrometry. (a) Chromatographic peak data for the peptides HGFLPR and KNIVTPR and their corresponding isotopically labeled forms (denoted by *) were generated from corresponding tandem mass spectra. Chromatographic peaks were generated by plotting the signal of the most intense fragment ion (y1–5 for HGFLPR and y1–6 for KNIVTPR) from tandem mass spectra filtered by the m/z of the precursor ion, with a seven-point Gaussian smoothing algorithm applied. (b) Collected tandem mass spectra were used to confirm peptide identity. Data were acquired with a mixture of the four peptides, each at a concentration of 1 nmol/L. (c) Dynamic range for MBP peptide quantification by tandem mass spectrometry. From 0.2 to 600 fmol of HGFLPR and KNIVTPR was quantified relative to 20 fmol of their respective isotopically labeled internal standards. The chromatographic peak ratio remained linear across four orders of magnitude, with a detection limit in the attomole range. Mean values are presented with the range denoted by error bars (n = 3).

Fig. 4

Quantifying the loss of the 18.6 kDa MBP isoform after TBI by isotope dilution mass spectrometry. In-gel clostripain digestion of the boxed gel slices was performed in a 7 nmol/L solution of the isotopically labeled HGFL*PR peptide (internal standard). Based on the ratio of endogenous HGFLPR to isotopically labeled standard, we calculated that the endogenous peptide (and associated intact MBP 18.6 kDa protein) was reduced 75-fold from (a) 510 fmol in naïve rat cortex (peak ratio of 37) to (b) 6.8 fmol in TBI rat cortex (peak ratio of 0.48).

Fig. 5

Gel electrophoresis separation of naïve and TBI samples. Prior to mass spectrometry analysis, we separated our brain samples by polyacrylamide gel electrophoresis to resolve MBP polypeptides measured against molecular mass markers (MKR). Slices were excised along each gel lane as indicated, from below an Ma of 30 to the bottom of the gel. The large band found in TBI lanes (Ma 12.5) is from hemoglobin that infiltrated the injured tissue by blood–brain barrier leakage.

Fig. 6

Quantitative profile of MBP in naïve and TBI samples. Profiles of HGFLPR (a) and KNIVTPR (b) abundance by Ma value for naïve and TBI rat cortex, overlaid with molecular mass labels demarking the correlating MBP isoforms and proteolytic fragments derived from data in Fig. 4. Mean values are presented with the measured range denoted by error bars (n = 3).

Fig. 7

Profile of MBP isoforms and proteolytic fragments in naïve and TBI cortex samples. Shown are profiles of the variably spliced exon sequence-specific peptides for naïve (a) and TBI (b) samples. Chromatographic peaks were generated for the most intense fragment ion of each peptide. Peak area means for each peptide (n = 3) were normalized across all Ma values. The presence of a peptide (and correlating exon-coded sequence) was determined when the normalized area at a given Ma was greater than three times the minimum for that peptide. Molecular mass labels demarking the identified MBP isoforms and proteolytic fragments are shown for each gel slice per the pattern of exon-specific peptides and given Ma value. ‘F12.6 kDa’ indicates a truncated 12.6 kDa fragment and ‘D4.3 kDa’ indicates a dimer of the 4.3 kDa fragment.

Fig. 8

Quantification of a calpain-induced MBP fragment in CSF of TBI rats. (a) 20 µL of CSF from naïve and TBI rats (pooled, n = 7 each) were resolved by gel electrophoresis. The KNIVTPR calpain-cleavage selective MBP fragment was detected in TBI and not in naïve for the indicated gel slices at an Ma value of 11. (b) The chromatographic peak ratio of endogenous KNIVTPR to isotopically labeled internal standard (KNIV*TPR) was 0.35, and when multiplied by 18.6 fmol of standard would denote a nominal 6.4 fmol of endogenous peptide with one-tenth of the sample analyzed. (c) Detection of the endogenous peptide was confirmed by the tandem mass spectrum, shown to match that of the isotopically labeled peptide standard (shifted by 5 m/z).