Combination of Gas-Phase Fractionation and MS3 Acquisition Modes for Relative Protein Quantification with Isobaric Tagging

Relative quantification of peptides and proteins with isobaric tags such as iTRAQ or TMT is commonly used in comparative quantitative proteomics based on tandem mass spectrometry (MS/MS). Nonetheless, isobaric tagging inherently suffers from the cofragmentation/interference phenomenon that may compromise the quality of the quantitative data. An MS3 acquisition mode has been recently proposed to address this issue. Because of the additional ion isolation and fragmentation step, the MS3 acquisition mode significantly alleviates this interference effect. However, MS3 acquisition exhibits a lower sensitivity and a higher duty cycle, both of which reduce the number of identified and quantified proteins. In the present study, we evaluated the combination of gas-phase fractionation (GPF) and MS3 acquisition modes to optimize both identification and quantification of tryptic peptides labeled with TMT using a hybrid ion trap-orbitrap (LTQ-OT) instrument. An interference model was used where TMT-labeled human plasma proteolytic digests were spiked with TMT-labeled E. coil proteolytic digests. When combined with GPF, the MS3 acquisition mode was compared with MS2 modes such as high-energy collision dissociation (HCD) and combined collision-induced dissociation (CID)/HCD. We demonstrated the benefit of using both GPF and MS3 to analyze tryptic peptides labeled with TMT in terms of quantification precision and accuracy as well as proteome coverage. We further explored parameters such as the influence of automatic gain control and additional MS3 scans. The TMT-GPF-MS3 workflow was shown to be a powerful alternative for quantitative proteomic studies that offers improved identification/quantification accuracy and enhanced proteome coverage without the need for extensive sample fractionation before MS analysis.

In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry

Luca Fornelli, Hubert Girault, Yu Lu, Sasa Miladinovic, Krzysztof Marek Piech, Yury Tsybin

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

2012

Extended Bottom-Up Proteomics with Secreted Aspartic Protease Sap9

Unige Anna Laskay, Kristina Srzentic, Yury Tsybin

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7 kDa on average) peptides in bottom-up proteomics to about twice longer (similar to 3.2 kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, similar to 1 h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH 5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7 kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. Biological significance Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (

Elsevier Science Bv2014

New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

Proteomics has been one of the main projects challenging biological and analytical chemists for many years. The separation, identification and quantification of all the proteins expressed within biological systems remain the main objectives of proteomics. Due to sample complexity, the development of fractionation, separation, purification and detection techniques that possess appropriate resolution to separate a large number of proteins, as well as being sensitive and fast enough for high throughput protein analysis are required. The objective of this thesis was to develop new separation strategies for protein/peptide fractionation using gel electrophoresis and its further detection by mass spectrometry analysis. A multi-electrode set up based on OFFGEL electrophoresis was developed. The objective was to provide a more efficient application of the electric field for fast and improved protein separation. The results showed that using a multi-electrode setup provides not only a higher protein separation but also better protein collection efficiency. Electrophoretic separation using an ultra narrow pH gradient (UNPG) gel was adapted for a three-well OFFGEL device for fast sample purification and desalting. Purification of an E.coli extract was applied to demonstrate that electrophoretic separation with UNPG gels provides an efficient strategy for fast purification of complex biological samples and can be utilized as a preparative technique in proteomics. UNPG gels were also used to separate charge molecules taking place in a new electro-elution device. The molecules were washed from the gel surface by an aqueous buffer and collected for further analysis by mass spectrometry. The electroelution device provides a fast approach that avoids time-consuming steps of extraction from the polyacrylamide gel. An electrostatic spray ionization (ESTASI) mass spectrometry technique was developed in our group to ionize sample solutions on different substrates. ESTASI has been here coupled to isoelectric focusing and demonstrated to be a powerful tool, which can improve the detection sensitivity compared to standard visualizing protocols. Paper-ESTASI-MS was developed and applied for rapid perfume analysis of six authentic fragrances as a high throughput and sample preparation-free method. ESTASI was also applied for quantitative analysis of caffeine in different beverages using a standard addition strip. The results were compared to classical standard addition methods for MS or LC. It was shown that the strip strategy could be utilized for fast and accurate food analysis and quality control. Desalting and direct analysis of samples from ZipTip by ESTASI-MS has been demonstrated.

EPFL2014

New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

EPFL2014