abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

Abstract

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the anti-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry.

The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

“…TAP-MS analyses of NS5A-associated protein complexes has been performed as previously described ( Pichlmair et al, 2012 ). The same samples were re-analyzed by liquid chromatography mass spectrometry (LCMS) on a hybrid linear trap quadrupole (LTQ) Orbitrap Velos (ThermoFisher Scientific) coupled to an Agilent 1200 series HPLC (Agilent technologies) as previously described ( Huber et al, 2014 ). The list of all identified proteins was uploaded to the ‘Contaminant Repository for Affinity Purification-Mass spectrometry data (CRAPome) repository’ ( www.crapome.org ) and filtered against a set of 17 negative control samples.…”

Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

“…Protein can also be concentrated and desalted in few steps. FASP can be combined with other methods, such as with isobaric tags for relative and absolute quantitation (iTRAQ) [ 147 ], a multiplexed approach to protein quantitation, and antibody affinity selection [ 148 ].…”

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

“…FASP‐assisted tryptic digestion was also performed by Huber et al. to affinity‐purified protein complexes or termed as affinity‐based FASP prior to LC‐MS and by Chen et al. to proteomes of endometrial tissue from midsecretory and proliferative phases of age‐matched women prior to nano‐LC‐MS/MS.…”