Novel silent albumin variant (191Ala → Thr) detected by TOF MS of whole plasma

• Rapid analysis of whole plasma by TOF MS reveals qualitative changes in serum albumin.
• Structural changes reflect mutations and circulatory modifications of the protein.
• Minute changes in whole protein mass were confirmed by DNA sequencing.
• New silent − 30 Da variant detected

BackgroundSerum protein electrophoresis occasionally reveals multiple albumin bands referred to as bis- or alloalbuminaemia, and whilst the condition can be inherited it may also be acquired.MethodsWe present a new high resolution approach to the investigation of qualitative changes in albumin structure. The on-line reverse phase time-of-flight mass spectrometry procedure (TOF MS) elaborated here requires < 0.2 μl of plasma and takes ~ 10 min to perform. Two plasma samples with classical bisalbuminaemia were used to verify the efficacy of the procedure for detecting genetic variants. When a novel mutation was detected, mass mapping of native unreduced albumin was used to pinpoint its location and inform targeted DNA sequencing of the albumin gene.ResultsNormal serum albumin showed its expected major isoform at 66,439 Da and the electrophoretic variants showed co-equal expression of additional components at − 14 and + 744 Da respectively. Surprisingly, one of the supposed controls showed paired albumin peaks at 66,439 and 66,469 Da and this 30 Da increase in mass was localised to between Arg114 and Arg197 and confirmed as being due to a novel 191Ala → Thr (+ 30 Da) substitution through DNA sequencing.ConclusionsThe electrospray TOF MS approach developed here provides a rapid, sensitive and extremely precise method of revealing minute changes in albumin primary structure.