RESUMEN
Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale.
Asunto(s)
Biopolímeros/química , Cromatografía en Gel/métodos , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Conformación ProteicaRESUMEN
New technology was used to identify traces of a commercial barrier/spermicide in evidence from a case of a man accused of rape of a minor. Examination of vaginal swabs performed by another laboratory had been negative for seminal fluid or other sources of DNA from the suspect and we were asked to examine the remaining swabs for any traces that might have originated from the commercial product. Encare consists of vaginal inserts having a suppository-like shape. They contain the spermicide, nonoxynol-9, in a matrix consisting of approximately two parts polyethylene glycol (PEG) 1000 to one part PEG 1450, plus minor inorganic components added to produce foaming. Portions of the cotton from vaginal swabs from the victim and penile swabs from the suspect were extracted with methanol and subsequently examined by desorption ionization on silicon time-of-flight mass spectrometry (DIOS TOF MS). Low levels of PEG in the same mass range as Encare were found on two separate vaginal swabs from the victim and one penile swab from the suspect. Subsequent to these findings, the suspect (through his attorneys) provided us with a sample of SLIP Plus, a commercial sexual lubricant that also contains nonoxynol-9. Traces of PEG in the same mass range as Encare were found in this sample, while no PEG was found in a sealed sample of SLIP Plus provided by the manufacturer. At trial the suspect's attorneys stipulated that their client had added some Encare to the SLIP Plus sample he had provided.