The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.
An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.
A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.
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