2D-PAGE Coupled to Mass Spectrometry for Proteomic Analysis of Human, Microbial and Plant Samples

In a typical proteomic experiment, proteins of interest are digested with enzymes for identification by mass spectrometric analysis. Trypsin is the most commonly used protease in proteomic analysis and displays good activity with in-gel digestion, as well as with in-solution digestion.
Mass spectrometers consist of an ion source that converts analyte molecules into gas-phase ions, a mass analyzer that separates ionized analytes on the basis of m/z ratio, and a detector that records the number of ions at each m/z value. Regarding ion sources, matrix-assisted laser desorption/ionization (MALDI) [48] and electrospray ionization (ESI) [49] ionization are central in proteomics.
MALDI is generally more salt-tolerant than ESI and it is typically coupled to time-of-flight (TOF) analyzers. For successful MALDI analysis, the key point is the proper choice of matrix and sample deposition method, to achieve the highest possible sensitivity and accuracy. The most popular matrices for proteomic applications include α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA) and 2,5-dihydrobenzoic acid (DHB). Particularly, MALDI mass spectrometry, thanks to its speed, sensitivity, accuracy, satisfactory tolerance to impurities and ease of automation, allows for protein identification by the set of measured proteolytic peptide masses. This process is known as peptide mass fingerprinting (PMF) and Mascot is among the most used algorithms in this identification approach [50]. Briefly, the experimental mass profile is matched against those generated in silico from the protein sequences in the database using the same enzyme cleavage sites. Unfortunately, the lack of sequence data makes identification by PMF ambiguous. The recent introduction of LC-MALDI technology offered a sophisticated and efficient mean of sample purification and separation combining the advantages of capillary liquid chromatography (LC) with the sensitivity and accuracy of MALDI-MS; in fact in the newest instruments, peptides eluted from the column are directly spotted onto target plate by robot machines [51].
ESI, instead, is often coupled to ion trap (IT) or quadrupole (Q) analyzers as this combination enables efficient peptide sequencing by induced fragmentation (MS/MS). In an ESI ion source the ionization process occurs under atmospheric pressure and the sample is introduced as liquid, therefore electrospray may be coupled directly to liquid chromatography systems. In particular, reversed-phase liquid chromatography coupled to tandem mass spectrometry (RP-LC-MS/MS) is a well established and commonly used procedure for identification of the in-gel digested proteins. Furthermore, newly introduced nano HPLC combined with ESI-MS/MS has emerged among the most widely used and the most powerful tools for proteomic research. Indeed, miniaturizing the HPLC separation column inner diameter improves electrospray sensitivity, because the concentration of equally abundant analytes in the LC mobile phase is proportional to the square of the column internal diameter. Moreover, because the sample must be loaded at flow rates of 200 nL/min, the sample volume to be injected onto a conventional nano HPLC system is usually only 1-2 μL. This approach usually requires an additional salt removal step during peptides preparation. In fact, a disposable C18 RP extraction tip, to remove insoluble particles and salts, and to preconcentrate peptides has been introduced [52]. For similar reasons, commercially available capillary chromatography systems include trapping pre-columns, where the sample is purified, desalted and pre-concentrated prior to injection onto the capillary column. In proteomic applications, the positive ion mode is mostly used, in which ionization is based on protonation of the analyte molecules, therefore the sample is often acidified, e.g. with formic or trifluoroacetic (TFA) acids, to facilitate ion formation.
Very recently, a new ionization technique was devised termed desorption electrospray ionization (DESI). Here the charged droplets of solvent are sprayed onto the analyzed object, so that molecules present on its surface are ionized [53]. DESI can be applied to solids, liquids (including complex biological samples) and adsorbed gases. Importantly, DESI apparently does not require sophisticated sample pre-treatment and tissue sections could be directly analyzed.