Identification of Multiple Splice and Charge Variants of LSP1 in WEHI-231 Cells and Primary B Cells

To detect ligand-induced changes in B-cell phosphoproteins, B cells were labeled with radioactive phosphate and analyzed by two-dimensional (2-D) gel electrophoresis. Resting WEHI-231 and primary B cells were lysed in isoelectric focusing (IEF) sample buffer and proteins were resolved by 2-D electrophoresis. Images of the SYPRO Ruby-stained gels were obtained using a 2-D Image Master fluorescence scanner and analyzed with Progenesis software to detect spots. The spot maps were filtered to remove artifactual spots caused by the presence of particulate material from the SYPRO Ruby stain. A total of 1208 and 1168 spots were detected in WEHI-231 and B cells, respectively, using a pH 4 to 7 gradient for the IEF dimension and a 12% acrylamide gel for second dimension SDS-PAGE. Although the pattern of protein spots in WEHI-231 and primary B cells were similar, there were clear differences between the two cell types (Fig. 1, panels A and B).

Fig. 1.Resolution of multiple charge variants of LSP1 by 2-D gel electrophoresis. Resting WEHI-231 cells and primary murine B cells were harvested and lysed using the preparative 2-D gel procedure. 500 mg of WEHI-231 lysate (panel A) or 750 mg of B-cell lysate (panel B) were resolved by 2-D gel electrophoresis using pH 4 to 7 gradient gels for the first dimension and 12% SDS gels for the second dimension. Following electrophoresis, the gels were fixed, stained with SYPRO Ruby, and scanned in a fluorescence imager. Panels C and D show a blown-up view of the region of the 2-D gels, outlined by boxes in panels A and B, respectively. The letters a through c in panels C and D refer to reference spots that had similar mobility in both the WEHI-231 and B-cell samples. Spots labeled L3 through L7 and S2 through S6 in panels C and D were identified as being derived from the long (L) and short (S) splice variants of LSP1 (GI:1346470; AfCS ID A002813).

We decided to focus on a prominent set of closely migrating phosphoproteins identified in both the WEHI-231 B lymphoma cell line and primary B cells. These prominent phosphoproteins appeared as a series of moderately abundant SYPRO Ruby-stained spots with isoelectric points between 4.4 and 4.6, and apparent molecular masses of 50- to 52-kDa. The mobilities of these spots (multiple charge variants with similar molecular weight) were consistent with the presence of differentially phosphorylated forms of single proteins. Two rows of spots (L3-L7, S2-S6) that differed by 2 to 3 kDa in apparent molecular mass were detected in both WEHI-231 and B cells (Fig. 1, panels C and D). The lower row of spots (S2-S6) was much less abundant in WEHI-231 than in B cells. The two series of spots had essentially identical isoelectric points and apparent molecular weights in the two cell types. Preparative-scale 2-D electrophoresis was then combined with mass spectrometry to identify these proteins.

Fig. 2. Identification of the long and short splice variants of LSP1 by mass spectrometry. Spots L5 and S5 from preparative 2-D gels of B cells were excised and digested with trypsin. The masses of the tryptic peptides were determined by oMALDI-QqTOF mass spectrometry. Panel A shows the mass spectrum obtained from spot L5, and panel B shows the mass spectrum obtained from spot S5. The masses of several peptides derived from murine LSP1 that were identical between the two spots are indicated. The two peptides marked with asterisks designate peptides that were unique to spots L5 and S5, in panels A and B, respectively. These peptides uniquely identify spots L5 and S5 as the long and short variants of LSP1.

Spots designated L3 to L7 and S2 to S6 in Fig. 1 were excised from preparative 2-D gels of both WEHI-231 and primary B cells and digested with trypsin. The mass spectra (MS) of the tryptic peptides and the fragments resulting from collision-activated dissociation (MS/MS) of the peptides were determined with an Applied Biosystems QSTAR Pulsar mass spectrometer equipped with a matrix-associated laser desorption ionization (MALDI) source. The MS and MS/MS data were used to search the NCBI non-redundant database using Knexus software. LSP1 was identified as the only, or the major, protein present in each of the spots analyzed (GI:1346470; AfCS ID A002813). Fig. 2 shows the MS of B-cell spots L5 and S5 as examples. Most of the MS peaks were identical in the two samples. However, a peptide with a mass-to-charge ratio (m/z) = 2107.96 was present in spot L5 but not spot S5. Conversely, a peptide with m/z = 1842.07 was present in S5, but not in L5. Comparisons of the masses of the parent peptides and fragments generated by MS/MS with the LSP1 sequence showed that these peaks corresponded to unique tryptic peptides generated from either the long (L5) or short (S5) splice variants of LSP1. The peak with m/z = 2107.96 in spot L5 was the peptide EPDPEDAVGGSGEAEEHLIR. The peak with m/z=1842.07 from spot S5 was the peptide EPDPEDAVGGSGEAEEVR. The difference between these peptides is consistent with the insertion of amino acids HLIRHQ into the long splice variant of LSP1 as reported by Matsumoto et al. (7). The same unique peptides were identified in each of the spots labeled L and S, respectively, in Fig. 1. The spots corresponding to the short splice variants in both WEHI-231 and B cells were shifted to more acidic isoelectric points relative to the long variants, consistent with the loss of a positive charge due to the absence of the arginine residue in the HLIRHQ peptide. Apart from the unique peptides present in the long and short variants, no other differences were detected in the peptide mass fingerprints of the 10 LSP1 spots analyzed from WEHI-231 and B cells. The different forms of LSP1 resolved in the IEF dimension were therefore due to differently charged forms of the same proteins. These observations were consistent with the presence of populations of LSP1 containing different amounts of phosphate.