Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain

Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new...

Case report: Interference from isatuximab on serum protein electrophoresis prevented demonstration of complete remission in a myeloma patient

Multiple myeloma is a haematological cancer caused by malignant plasma cells in the bone marrow that can result in organ dysfunction and death. Recent novel treatments have contributed to improved survival rates, including monoclonal antibody therapies that target the CD38 protein on the surface of plasma cells. Anti-CD38 therapies are IgG kappa monoclonal antibodies that are given in doses high enough for the drug to be visible on serum protein electrophoresis as a small paraprotein. We present a case where isatuximab, the most recent anti-CD38 monoclonal antibody to be approved for treatment of myeloma, obscured the patient’s paraprotein on gel immunofixation, so that complete remission could not be demonstrated. This was resolved using the isatuximab Hydrashift assay. The interference on gel immunofixation was unexpected because isatuximab migrated in a position distinct from the patient’s paraprotein on capillary zone electrophoresis. We demonstrate the surprising finding that isatuximab migrates in a different position on gel electrophoresis compared to capillary zone electrophoresis. It is vital that laboratories are aware of the possible interference on electrophoresis from anti-CD38 monoclonal antibody therapies, and are able to recognise these drugs on protein electrophoresis. The difference in isatuximab’s electrophoretic mobility on capillary and gel protein electrophoresis makes this particularly challenging. Laboratories should have a strategy for alternative analyses in the event that the drugs interfere with assessment of the patient’s paraprotein.

Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein

The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations. Graphical abstract

Unexpected Discovery of Hemoglobin D Iran Families in Punjab, Pakistan by using two different Screening Methods

Background: Hemoglobin D Iran is frequently misdiagnosed as Hb E or Hb D Punjab if only one method of screening is used. The objective of our study was to highlight the importance of using two different screening techniques in diagnosis of a hemoglobin variant, Hb D Iran in our case. Hematological parameters of heterozygous Hb D Iran and compound heterozygous β/Hb D Iran were also compared. Methods: A descriptive study was carried out on results of 52,379 subjects which were part of thalassemia extended family cascade screening from 36 districts of Punjab from October 2019-March 2021. Cases of Hb D Punjab and Hb E were run on both CE-HPLC (cation exchange-high performance liquid chromatography) and CZE (capillary zone electrophoresis). Resulting Hb D Iran cases were confirmed by ARMS-PCR (Amplification refractory mutation system-polymerase chain reaction). Results: Forty cases of Hb D Iran were detected out of 160 initially suspected Hb D Punjab cases and 126 Hb E cases. Diagnosis was confirmed by molecular analysis. Statistical significance was found between RBC count, MCV, MCH, Hb F and diagnosis of “heterozygous Hb D Iran” and “compound heterozygous for β/ Hb D Iran”. Conclusion: Hb D Iran can be easily missed and misdiagnosed as Hb E or Hb D Punjab, if two screening methods are not used. This maybe a reason why Hb D Iran remains unreported in our region. CBC and HPLC indices can also be suggestive if a case is of heterozygous D Iran or compound heterozygous β/Hb D Iran. Keywords: Hb D Iran, Hb E, Hb D Punjab, Cation exchange High performance liquid chromatography, Capillary zone electrophoresis