Eliminating the Interferences from TRIS Buffer and SDS in Protein Analysis by Fused-Droplet Electrospray Ionization Mass Spectrometry

Abstract

Multiply charged protein ions were detected from the solutions containing a high concentration of tris(hydroxymethyl) aminomethane buffer (TRIS) and sodium dodecyl sulfate (SDS) using fused-droplet electrospray ionization mass spectrometry (FD-ESI/MS). The sample aerosols were generated at ambient temperature with a pneumatic nebulizer commonly used to produce sample aerosols in an atmospheric pressure chemical ionization (APCI) source. The aerosols were carried by nitrogen gas to the tip of a capillary where charged methanol droplets had been continuously generated by electrospraying an acidic methanol solution. The neutral sample aerosols then fused with the charged methanol droplets and electrospray ionization proceeded from the newly formed fused droplets to generate multiply charged protein ions. Because of its low solubility in methanol, TRIS molecules (concentration as high as 1 M) were efficiently excluded from the newly formed droplets and the protein ion signals were detected and observed in the mass spectra. To remove the interferences from SDS, equal moles of positively charged cetyltrimethylammonium bromide (CTAB) was added into the SDS containing sample solution to form the dodecyl sulfate-cetyltrimethylammonium ion pair (DS-CTA). The DS-CTA ion pair has a low polarity and solubility in methanol and is excluded from the fused droplet. Protein ions were still detected from the solution containing 10(-2) M of SDS.

What can we learn from ambient ionization techniques?

“…For example, some authors are adamant about a differentiation between EESI and SESI, a method and acronym originally introduced by Hill and coworkers [6]: for example, Martinez-Lozano and co-authors [137] argue that in cases where breath is analyzed, one should use the term SESI, whereas in our work on breath analysis, the acronym EESI was always used [5,138] to indicate that breath is in fact an aerosol [139]. Yet other workers refer to this process as FD-ESI [134,140], and there are close similarities to a method called "remote reagent chemical ionization," which has appeared in the patent literature [141]. We suggest that these differences are not important enough to justify the use of more than one acronym.…”

Generation of multiply charged peptides and proteins by radio frequency acoustic desorption and ionization for mass spectrometric detection

“…By constantly electrospraying parallel to the sample surface (above the analyte spot) during the desorption event, desorbed species interacted with the ESI droplets for ionization. This is analogous to ionization in fused droplet-electrospray ionization (FD-ESI) [12,13] and extractive electrospray ionization (EESI) [14 -18] due to neutral interaction with an ESI plume. Previous reports demonstrated that postdesorption ionization offers the advantage of withstanding high salt tolerances [12].…”

“…Several other techniques are mentioned below as they utilize postdesorption ionization (as in RADIO). Secondary electrospray ionization (SESI) [8 -11] involves the gas-phase interaction of charged ESI droplets with neutral sample molecules for analysis by ion mobility spectrometry (IMS) or MS. Fused droplet electrospray ionization (FD-ESI) [12,13] aerosolizes the sample solution via a nebulizer for interaction with a highly charged acidic methanol solution and reduces interferences from buffers and complex mixtures. Extractive electrospray ionization (EESI) [14 -18] employs two nebulizing sprayers, one with ESI solvents and a second containing the analyte of interest for a liquidliquid extraction process to reduce interferences from complex mixtures.…”

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

“…Thus prior to the addition of trypsin, a buffer is added (usually 50 mM triethyl ammonium bicarbonate (tABC) or 12.5 mM ammonium bicarbonate (ABC) buffer (López-Ferrer et al 2006) to provide an optimal pH for the enzymatic cleavage. A 2-amino-2-hydroxymethylwww.intechopen.com propane-1,3-diol (Tris) buffer may also be used for this purpose, but it should be taken into consideration that the Tris buffer is incompatible with the down stream MS analysis, such as MALDI and ESI-MS, and needs to be depleted through solid phase extraction (SPE) or ZipTips prior to such (Shieh et al 2005;Sigma-Aldrich 2011). Information about the enzyme to protein ratio needed for digestion of a protein sample is crucial to ensure an enzyme amount sufficient to perform the digestion, but not too high resulting in autolysis products from the trypsin used.…”