Crystalglobulinemia (cryocrystalglobulinemia) is a rare complication of multiple myeloma. Crystallization of immunoglobulin in blood circulation causes systemic vasculopathy especially in skin and kidney. We found a rare case of crystalglobulinemia in which the light chain was N-glycosylated. The abnormal N-glycosylation was primarily detected as the molecular mass shift on SDS-PAGE by PNGase F treatment. The cryocrystalglobulin was shown to be composed of 55-kDa heavy and 32-kDa light chains on SDS-PAGE. However, the apparent molecular masses of them shifted to 51 kDa and 28 kDa, respectively by PNGase-F treatment. The cryocrystalglobulin was identified as an IgG κ type by peptide mass fingerprinting. The N-glycans on the κ light chain were assigned to non-fucosylated biantennary oligosaccharides and their bisected forms by MALDI-TOF MS/MS analysis of glycopeptides. Sialylation of the abnormal N-glycans was suggested by linear-mode MS and confirmed by HPLC analysis. The N-glycosylation consensus Asn (Asn-Xxx-Ser/Thr) was found in the glycopeptide at the N-glycosylation site determined as “EIVMTQSPANLSVLPGER” by MALDI-TOF MS/MS, in which the consensus Asn (N) was converted to Asp (D) in the enzymatically deglycosylated peptide.

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We measured the urinary excretion of retinol-binding protein (RBP), β₂-microglobulin (β₂-m), albumin, and N-acetyl-β-D-glucosaminidase as well as serum glucose, RBP, β₂-m, hemoglobin A1c, and creatinine in type 2 diabetes mellitus 158 patients. In urinary RBP, there was a significant difference between the patients who had nephropathy and those without complications. In the cases with nephropathy, urinary RBP had a significantly high value, but no significant difference was observed in the case with retinopathy. There were no significant correlations between urinary RBP and serum RBP. The index of correlation was 0.351.
 According to western blot, there were 11, 6, and 6 bands for serum RBP, urinary RBP, and urinary transthyretin (TTR), respectively. The main bands for all three had molecular mass (MM) of 150 kDa. RBP combined with TTR is thought not to be able to pass through kidney glomeruli due to its high MM. The main band of urinary RBP is free RBP according to previous reports. Nevertheless, the main band had a MM of 150 kDa and represented combined TTR. This result contradicts previous reports about urinary RBP.
 On the other hand, for urinary RBP4, the only band was of 150 kDa for cases without retinopathy, but for the cases with retinopathy, a sub-band was seen at 50 kDa and an extremely light-colored band was seen at 55 kDa, which suggests the possibility of early detection of retinopathy by examining these bands.

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Serum provides a link between many human organs, tissues, and cells, and is one of the most informative body fluids. However, the presence of 22 abundant proteins and a large dynamic range of numerous other proteins make quantitative analysis of low abundance proteins challenging. Here, we describe simple and easy to use pretreatment techniques for serum combined with high abundant protein removal and reverse-phase high-performance liquid chromatography (RP-HPLC) separation, each step of which was optimized to minimize the loss of proteins and increase reproducibility. This method can be used to discover disease-specific biomarker proteins in concentrations of the ng/mL range, and furthermore, be used as a base strategy to comparatively analyze the deep proteome in the pg/mL range.

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