iFASP: Combining Isobaric Mass Tagging with Filter-Aided Sample Preparation

Abstract

Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging) since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced Filter-Aided Sample Preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.

Kidney Response to Heart Failure: Proteomic Analysis of Cardiorenal Syndrome

“…The iTRAQ labelling was integrated into the FASP workflow as described [20]. In total three iTRAQ analyses were performed according the same scheme.…”

endoProteoFASP: A novel FASP approach to profile salivary peptidome and disclose salivary proteases

“…The dual use of a 30 kDa cut-off filter as a sample cleaner device and as a "proteomic reactor" allowed proper separation of peptides and, at the same time, an increase in the activity of soluble salivary proteases. Thus, we provided the proof-of-concept of a novel peptidomic approach, the endoProteo-FASP, to understand the dynamics of the salivary peptidome, making the most of saliva's own protease pool and avoiding the use of synthetic peptides and exogenous proteases, as previously reported in the literature [12,[15][16][17][18][19][20][21][22]27]. Additionally, our work confirmed the utility of the combination of the ammonium bicarbonate extraction method with sample filtration in order to enrich salivary peptides, as reported previously by our group [28].…”

“…An example of other adaptations is provided by Wiśniewski and Mann, which developed a multienzyme digestion FASP (MED-FASP) approach that increases the proteome coverage, due to consecutive proteolysis of the retained material with two or more proteases [16]. The combination of FASP with isobaric mass tagging (iFASP) was also proven possible with higher peptide labeling efficiency than the conventional protocols provided by manufacturers [17]. The versatility of FASP is notorious in numerous studies, which describe protocol adaptations to attain different purposes.…”

Advances in proteomics for production strain analysis

“…The FASP technique, first described in 2009 [49] has been adapted for improved proteolytic digestion (eFASP) [50] and for compatibility with chemical labelling strategies for analysis of proteins (iFASP) [51] or affinity purified protein complexes (abFASP) [52]. Protein-protein interactions (PPI) is a growth area in proteomic analysis, particularly since proteins typically function in complexes which are temporally and spatially dynamic within the cell [53] and analysis of PPI has value in system based network modelling [54].…”