Computational protocol: Multiple marker abundance profiling: combining selected reaction monitoring and data‐dependent acquisition for rapid estimation of organelle abundance in subcellular samples

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Protocol publication

[…] For CSCs and 4‐week rosette samples (five replicates each), MS/MS data were acquired from about 1 μg peptides with a nano‐ESI‐Q‐TOF system (TripleTOF® 5600 System, SCIEX) coupled to an Eksigent nano LC system (SCIEX). Peptides were separated on a Pepmap100 μ‐guard column (Dionex) via a Famos Autosampler (Dionex) and washed for 10 min with Buffer A (2% acetonitrile, 0.1% formic acid) flowing at 15 μl min−1. Peptides were eluted onto an Acclaim Pepmap100 C18 column (75 μm × 150 mm, 300 nl min−1 flow rate; Dionex) and into the TripleTOF 5600 via a gradient consisting of initial starting condition of 5% buffer B (98% acetonitrile, 0.1% formic acid) increasing B to 35% B over 60 min. Subsequently, B was increased to 90% over 3 min and held for 15 min followed by a ramp back down to 5% B over 3 min where it was held for 15 min to re‐equilibrate the column to the original condition. Peptides were introduced to the mass spectrometer via a Nanospray III source (SCIEX) with a nano‐tip emitter (New Objective) operating in positive‐ion mode (2400 V). The data were acquired with Analyst TF 1.5.1 operating in information‐dependent acquisition mode, whereby after a 250‐msec scan the 20 most intense ions (charge states 2–5) within 400–1600 m/z mass range above a threshold of 150 counts were selected for MS/MS analysis. MS/MS spectra were collected using TOF Resolution Mode: High Resolution with the quadrupole set to UNIT resolution and rolling collision energy to optimize fragmentation. MS/MS spectra were scanned from 100 to 1600 m/z, and were collected for a total accumulation time of 50 ms. selected precursor ions were excluded for 16 sec following MS/MS acquisition. The raw data were processed with the ProteinPilot Software package v.4.0 (SCIEX) and matched with the Paragon Algorithm against Arabidopsis proteins (TAIR10; Lamesch et al., ). The Paragon Method (Shilov et al., ) employed standard settings with the instrument set as ‘TripleTOF 5600’ resulting in initial search parameters of 0.05 Da (MS) and 0.1 Da (MS/MS). The detected protein threshold was set at 99% [Unused ProtScore (Conf) > 2.0] and a Thorough ID was applied for the Search Effort. The data processing and matching by ProteinPilot results in recalibration of data, which were subsequently exported as MGF Peaklist(s) for HC‐data matching. These raw data for the whole plant (n = 3) and CSCs (n = 3) are available at PRIDE (Project https://doi.org/10.6019/pxd005408).For Arabidopsis low/high‐light samples, analysis was undertaken with about 1 μg protein and performed with a Q‐Exactive+ (Thermo Fisher Scientific) with a nanoACQUITY UltraPerformance LC system (Waters), incorporating a C18 reverse phase column (Waters; 100 μm × 100 mm, 1.7 μm particle, BEH130C18, column temperature 40°C). Peptides were analysed over a 150‐min gradient using Buffer A (2% acetonitrile, 0.1% formic acid), 5% Buffer B (98% acetonitrile, 0.1% formic acid). Buffer B was increased from 2 to 10% over 2 min, to 40% over 110 min, then to 85% over 1 min, maintained at 85% for 10 min and equilibrated for 14 min with 2% buffer B. Peptides were eluted at a flow rate of 300 nl min−1. An MS survey scan was obtained for the m/z range 300–1600. MS/MS spectra were acquired using a top 15 method, where the top 15 ions in the MS spectra were subjected to high‐energy collisional dissociation. An isolation mass window of 2.0 m/z was used for the precursor ion selection, and normalized collision energy of 27% was used for fragmentation. A duration of 5 sec was used for the dynamic exclusion. An automatic gain control target of 1 000 000 for MS and 50 000 for MS/MS was used, while maximum IT for MS was 30 msec and MS/MS was 50 msec. The system employed a resolution of 70 000 for MS and 17 500 for MS/MS. Tandem mass spectra were extracted, charge state was deconvoluted, and raw data files were converted to MGF picklists by Proteome Discoverer version 1.4 (Thermo Fisher Scientific). Data are available at PRIDE (Project https://doi.org/10.6019/pxd005408). For datasets 22–24 (Figure ), protoplasts were generated homogenized as in Parsons et al. (), clarified by centrifugation at 3000 g for 10 min, then processed as described in Christoforou et al. (), with the exception that in the second centrifugation step at 100 000 g, the supernatant was underlaid with a 25% iodixanol cushion. Spectral data are available in Table . [...] The MGF peaklists were each interrogated with the Mascot search engine version 2.3.02 (Matrix Science). For TripleTOF® 5600 System data, a peptide tolerance of ± 50 ppm and MS/MS tolerance of ± 0.100 Da and the instrument type was set to ESI‐QUAD‐TOF. For data produced on the Q‐Exactive+, a peptide tolerance of ± 10 ppm and MS/MS tolerance of ± 0.050 Da with the instrument type set to ESI‐FTICR. Shared search parameters included variable modification of oxidation (M); fixed modifications of carbamidomethyl (C); up to one missed cleavage for trypsin. All searches were performed against Arabidopsis proteins (TAIR10) and the common Repository of Adventitious Proteins (cRAP version 1.0, The Global Proteome Machine) comprising 35 393 proteins. Mascot search results were imported into Scaffold (v4.3.4, Proteome Software) with the following filters: peptide identifications greater than 95.0% probability by the Peptide Prophet algorithm (Keller et al., ) with Scaffold delta‐mass correction; protein identifications >99.0% probability; and protein identification containing at least one identified peptide. Scaffold was used to determine average SpC for each protein in a discrete experiment (4‐week rosettes, CSCs, high‐light and low‐light conditions) by loading each replicate as a BioSample with LFDR scoring (all instruments) and protein cluster analysis parameters selected. Proteins with fewer than three spectra were discarded from analyses. […]

Pipeline specifications

Software tools Analyst TF, ProteinPilot, Proteome Discoverer, Mascot Server
Databases TAIR
Application MS-based untargeted proteomics
Organisms Arabidopsis thaliana