Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues

Abstract

Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.

Stress-Induced Chloroplast Degradation in Arabidopsis Is Regulated via a Process Independent of Autophagy and Senescence-Associated Vacuoles

“…To elucidate the mechanism(s) by which CV disrupts chloroplasts, we identified potential CV-interacting proteins using coimmunoprecipitation (co-IP) and subsequent identification of interactors by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Smaczniak et al, 2012). Antibodies raised against HA were conjugated to magnetic beads, and these beads were used to immunoprecipitate CV-HA and its interacting proteins from total protein extracts obtained from DEX-treated transgenic plants expressing DEX-CV-HA (DEX-3 line).…”

The BRASSINOSTEROID INSENSITIVE1–LIKE3 Signalosome Complex Regulates Arabidopsis Root Development

“…Initial pull-down experiments using ProBRL3:BRL3-expressing seedlings were performed using the protocols described by Smaczniak et al (2012). Coimmunoprecipitation experiments were done in the same conditions as the IPs described above, but instead of 10 g of starting material, 5 g was used for homozygous lines coexpressing ProBRL3:BRL3-YFP and ProBAK1:BAK1-HA.…”

“…Three independent biological replicates were performed for each tagged receptor and wild-type complex purified in pairs. The resulting peptides were analyzed by LC/MS/ MS as previously described (Smaczniak et al, 2012). First, the ProBRL3:BRL3-YFP line was used.…”

“…As an alternative approach, we previously employed immunoprecipitation (IP) of the green fluorescent protein (GFP)-tagged SERK1 coreceptor (Karlova et al, 2006;Smaczniak et al, 2012). This resulted in the identification of BRI1 as well as BAK1, suggesting that at least in part, protein-protein interactions mirror the genetic evidence.…”

Stem cell organization in Arabidopsis : from embryos to roots

“…This methodology has been successfully used to identify complexes of multiple types of proteins, including transcription factors and other nuclear proteins Saiga et al, 2012), membrane associated proteins (Zwiewka et al, 2011), trans-membrane proteins (Zwiewka et al, 2011), and cytosolic proteins (Smaczniak et al, 2012). When considering IP-MS/ MS for the identification of interacting proteins to a protein of interest, it is important to keep in mind that this is not a saturated identification method.…”