Marina Galvani

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF)‐mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to “scrambled” disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human α‐ and β‐globin chains, which possess only one (α‐) or two (β‐) ‐SH groups. As a result, misplaced spots are to be found in the resulting two‐dimensional (2‐D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS‐PAGE step.

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.

All existing protocols for protein separation by two‐dimensional (2‐D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) to monitor protein alkylation by iodoacetamide over the period 0 – 24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached ∼70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2‐D gel analysis in particular and proteomics in general are discussed.

Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.

Although it is highly recommended that reduction and alkylation of free –SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA).The present matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free –SH group of Cys in proteins at alkaline pH values (pH 8.5–9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the –SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.

Effect of experimental conditions on the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ ionisation mass spectrometry.

Two mixtures of proteins having molecular weights in the range approximately 8-97 kDa were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). Part of our aim in this study is to gain more insight into the influence of the various experimental conditions on the overall quality of the acquired mass spectral data. Different protein extraction procedures, two staining agents, and extraction times, were among the parameters assessed. In terms of the overall quality of the acquired mass spectra and the speed of protein recovery, ultrasonic assisted passive elution, into a solvent mixture containing formic acid/acetonitrile/2-isopropanol/water, was found to be more efficient than other elution procedures. The higher resolution associated with the delayed extraction mode allowed the identification of a number of protein modifications, including multiple formylation provoked by formic acid, cysteine alkylation caused by unpolymerised acrylamide monomers, and complexation with the staining reagents. The detection of these modifications, however, was limited to proteins under 30 kDa. Analysis of a ubiquitin tryptic digest by reflectron MALDI time-of-flight (TOF) allowed reliable identification of a number of the formylation sites.

Protein alkylation by acrylamide, its N-substituted derivatives and cross-linkers and its relevance to proteomics: A matrix assisted laser desorption/ionization-time of flight-mass spectrometry study

The present review highlights some important alkylation pathways of proteins, as measured by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF)‐mass spectrometric analysis, engendered by acrylamide and a number of its derivatives, including N‐substituted acrylamides, cross‐linkers and Immobilines (the acrylamido weak acids and bases used to create immobilized pH gradients). The present data are of relevance in two‐dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the –SH group of proteins even when engaged in disulfide bridges. An order of reactivity is obtained for a series of cross‐linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, originating “pendant, unreacted ends”, which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species being the acidic compounds. When analyzing real‐life samples by two‐dimensional (2‐D) maps, like milk powders, a number of modifications can be detected by MALDI‐TOF mass spectra of eluted spots, including variable phosphorylation sites (up to nine) and lactosyl moieties. If, for eluting such spots, formic acid is used, MALDI‐TOF mass spectrometry (MS) reveals an incredible number of formylation sites, on Ser and Thr residues.

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

Proteins in a commercial milk powder have been separated by two-dimensional gel electrophoresis and analysed by matrix-assisted laser desorption ionisation mass spectrometry. The mass spectrometric analyses were conducted in two steps: analysis of the intact proteins following their passive extraction into a suitable solvent mixture and analysis in reflectron mode of in situ digests of a number of gel spots. The combination of the two methods allowed a reliable identification of a number of proteins, including nine caseins as well as certain protein modifications including single/multiple phosphorylation, lactose-protein conjugates and Coomassie Brilliant Blue adducts. Analyses of the intact proteins prior to their in situ digestion contributed to a more efficient and reliable consultation of protein databases.

Investigating the reaction of a number of gel electrophoresis cross-linkers with β-lactoglobulin by matrix assisted laser desorption/ionization-mass spectrometry.

A number of cross‐linkers that are commonly used in polyacrylamide gels have been incubated with bovine β‐lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization‐mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross‐linker incubated for 1 h with 50 pmol/μL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N′‐bisacrylylcystamine > bisacrylyl piperazine > N,N′‐methylenebisacrylamide >> N,N′‐diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post‐source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross‐linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross‐linkers. Investigation of solutions containing a cross‐linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.

Probing protein unfolding through monitoring cysteine alkylation by matrix-assisted laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation mass spectrometry was used to monitor interaction between three proteins and two basic Immobiline chemicals (pK 10.3 and pK >12) commonly used in immobilised pH gradients (IPG). For two of the investigated proteins, the observed alkylation channels of the cysteine residues exhibited unmistakable response to their gradual denaturation following treatment with different concentrations (0-8 M) of two commonly used denaturants, urea and guanidine hydrochloride. Our assessment for protein unfolding is based on the number and relative intensity of the alkylation channels, yet the present mass spectrometry data are in good agreement with data based on optical rotatory dispersion, in which another approach was used to assess protein unfolding. Whether the present simple, fast and specific mass spectrometry method can be developed as a probe for monitoring folding/unfolding of cysteine-containing proteins can only be demonstrated by generating similar data for a larger number of proteins.