Reactive-electrospray-assisted laser desorption/ionization for characterization of peptides and proteins.
Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS ( n ) to protein analysis and show that ELDI is capable of multistage MS to MS (4) for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce "reactive-ELDI", which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.
Peng
Ivory X
IX
Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095, USA.
Ogorzalek Loo
Rachel R
RR
Shiea
Jentaie
J
Loo
Joseph A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000378:metabolism; D004579:Electron Transport; D005740:Gases / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008970:Molecular Weight; D009930:Organic Chemicals / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D004220:Disulfides; D005740:Gases; D007328:Insulin; D009930:Organic Chemicals; D010455:Peptides; D011506:Proteins; D012997:Solvents; D005978:Glutathione; D004229:Dithiothreitol