Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels

Abstract

A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of a milk powder

“…Each spot was carefully cut, crushed and treated with 5 mL of an extraction solution (FAPH), 15 containing 50% formic acid, 25% acetonitrile, 15% isopropanol and 10% water, in an ultrasonic bath for 15 min. The sample was then centrifuged at 10000 rpm for 2 min and the supernatant was collected and loaded directly onto the MALDI sample plate, using the three layers method, as described in the mass spectrometry section.…”

“…For example, b-lactoglobulin which has M r % 18300 Da is observed here above 20000 Da, while casein proteins are observed around 40 kDa instead of %25 kDa. To gain more accurate M r values, the various spots were carefully cut, crushed and immersed in a solvent mixture (50% formic acid, 25% acetonitrile, 15% isopropanol, 10% water) 15 and ultrasonicated for 10 min. 2 mL of the resulting extract were mixed with an equal volume of sinapinic acid and subjected to MALDI-TOF analysis.…”

“…3 (a) which shows a single peak at m/z 18431. Such differences in resolution can be attributed to possible protein modifications such as acrylammide adducts [17][18][19] and formylation, 15,20,21 which are known to occur during the separation and extraction procedure. Despite such reduced resolution the data in the same figure highlight two known modifications: single and double lactose conjugates at m/z 18758 and 19090, respectively, and a single Coomassie Brilliant Blue adduct at m/z 19269.…”

Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis

“…The gel pieces were dried under vacuum, then incubated in 50 mL of formic acid/ACN/isopropanol/water (50/25/15/10 v/v/v/v) in an ultrasonic bath at 35 o C for 30 min [11]. The resulting solution was mixed 1:1 with 1% w/v cyano-4-hydroxycinnamic acid in 50% v/v ACN containing 0.1% w/v TFA and 1 mL spotted onto a stainless steel target and air-dried.…”

Azo‐group reduction during the matrix‐assisted laser desorption/ionization process in the presence of 2,5‐dihydroxybenzoic acid

“…For over 20 years, matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used to identify a wide range of analytes such as peptides,1 proteins,2 oligosaccharides3 and nucleic acids 4. Despite the extensive use of this type of MS, we still do not fully understood the physicochemical processes taking place after the absorption of laser energy in the crystal, or the following material desorption and expansion.…”