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ABSTRACT: E. coli proteins (300 ng) extracted from the sample were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 (Thermo Fisher Scientific) reversed-phase liquid chromatography (RPLC) separation system with a C2 column (60 cm length, CoAnn Inc.). In the RPLC system, phase A was water with 0.1% formic acid (FA), and phase B was 60% acetonitrile (ACN) and 15% isopropanol (IPA) with 0.1% FA. A 98-min gradient of mobile phase B (0-5 min 5%, 5-7 min for 5% to 35%, 7-10 min for 35% to 50%, 10-97 min for 50% to 80%, 97-98 min from 80% to 99%) was applied with a flow rate of 400 nL/min. E. coli proteins were analyzed using both DDA and DIA modes. In each mode, six runs were performed separately, with each targeting a specific m/z range within the MS1 scan: 720-800, 800-880, 880-960, 960-1040, 1040-1120, and 1120-1200 m/z. MS1 spectra were collected with a resolution of 240,000 (at 200 m/z), 4 microscans, an automatic gain control (AGC) target value of 1x106, and a maximum injection time of 200 ms. MS/MS spectra were obtained with a scan range of 400-2000 m/z, a resolution of 60,000 (at 200 m/z), 1 microscan, an AGC target value of 1x106, and a maximum injection time of 500 ms. Fragmentation was performed using higher-energy collisional dissociation (HCD) with 30% NCE. In the DDA runs, the top six precursor ions from each MS1 scan were isolated with a 3 m/z window for MS/MS analysis. The dynamic exclusion was set to 60 seconds. In the DIA runs, a 4 m/z isolation window was used, resulting in a total of 20 MS/MS spectra for each cycle. Three technical replicates were obtained for each experiment.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli K-12 (ncbitaxon:83333)

SUBMITTER: Xiaowen Liu

PROVIDER: MSV000094362 | MassIVE | Tue Mar 19 10:31:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:We highlight the leap in performance made possible with new instrumentation in the Thermo Scientific Orbitrap Astral™ mass spectrometer. Integrated alongside a high-resolution accurate mass (HRAM) Orbitrap analyzer, the instrument introduces a fast-switching quadrupole mass filter congruent with downstream speeds of ion detection, a dual-pressure ion processor cell which fragments ions in the high-pressure cell before they are moved to the low-pressure cell for further accumulation and orthogonal extraction to the Astral analyzer, and a high dynamic range detector. Altogether, ions can be manipulated in five stages of the MS at once (Orbitrap, ion routing multipole, two ion processor stages, Astral analyzer), allowing for a high degree of parallelization which enables MS/MS scan speeds of 200 Hz while HRAM MS1 full scans are synchronously acquired in the Orbitrap analyzer. To provide such fast scan speeds, the Astral analyzer is capable of single-ion detection sensitivity like a linear ion trap, all while enabling resolving powers of 80,000 at m/z 524. Here, in triplicate 7-min microflow active LC gradients on the Orbitrap Astral MS, we report 7,852 protein groups from 94,267 peptides on average. When employing 15-, 30-, and 60-min active, nano-LC gradients, triplicate experiments yield an average of 9,831, 10,411, and 10,645 unique protein groups from 195,612, 234,406, and 245,754 unique peptides, respectively. These nanoflow methods delivered a median sequence coverage of 38%, 42.4% and 44.4% across identified proteins for each respective gradient length. Our 30-min method delivered approximately 347 proteins per minute. Finally, we applied our 30-min active gradient method to analyze a CRISPR-Cas9 knockout (KO) of the mitochondrial gene MGME1 in human HAP1 cells. There, we quantified an average of 10,353 proteins across three technical replicates and find 449 significantly up- or down-regulated proteins relative to wild-type (WT) HAP1 proteins. Proteins from the same sample quantified using a 61-min active gradient Orbitrap Ascend DIA method show strong fold-change correlations to and gene ontology (GO) term agreement with our results.Building upon the immense progress of the mass spectrometry and proteomics communities of the past ten years, here we report alongside other recent works, that the one-hour human proteome is now within reach.

Project description:Serum samples were inactivated and sterilized at 56 ℃ for 30 min and processed as previous study with some modifications (Shen et al., 2020). Briefly, 4 μL serum from each sample was depleted using High Select Top-14 Abundant Protein Depletion MiniSpin Columns (Thermo Fisher Scientific, San Jose, USA). Then the elutes were denatured, reduced and alkylated to derive protein lysates. The solutions were diluted with 200 μL 100 mM triethylammonium bicarbonate (TEAB) followed by a double-step trypsinization (enzyme-to-substrate ratio kept at 1:40 in each step). 10% trifluoroacetic acid (TFA) was added to each sample to quench the enzymatic reaction. Digested peptides were cleaned-up with SOLAμ (Thermo Fisher Scientific, San Jose, USA) according to the manufacturer’s instructions and labeled by TMTpro 16 plex reagents (Thermo Fisher Scientific, San Jose, USA). 660 samples in total (633 serum samples and 27 technical replicates) were divided into 44 batches for labelling experiment, each batch containing fifteen samples and one pool. The labeled samples in each batch were combined and fractionated. Each sample was separated into 30 fractions and then consolidated into 10 fractions. Dried and re-dissolved fractions with 2% acetonitrile (ACN)/0.1% formic acid (FA) of MS grade. The re-dissolved peptides were analyzed with a U3000 HPLC system coupled to a Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany) in data-dependent acquisition (DDA) mode combined to a front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS). Peptides of each fraction were loaded onto a pre-column (3 μm, 100 Å, 20 mm*75 mm i.d.) and separated with a 75 min LC gradient at a flow rate of 300 nL/min (analytical column: 1.9 mm, 120 Å, 150 mm*75 mm i.d.; Buffer A: 2% ACN, 98% H2O containing 0.1% FA; Buffer B: 98% ACN, 2% H2O containing 0.1% FA). FAIMS was operated at two different CV, -48 V and -68 V, respectively. For MS acquisition, RF level was set at 50% and ion transfer tube temperature at 320 °C. The turbo-TMT mode was enabled. Scan range of MS1 was 350–1800 m/z. Resolutions were set to 60000 for MS1 and 30000 for MS2. Normalized AGC target was set at 300% for MS1 and 200% for MS2. The maximum injection time was set as 50 ms for MS1 and 86 ms for MS2. Dynamic exclusion was on and mass tolerance was set to ±10 ppm. Intensity threshold was set at 20000 for MS1, and HCD collision energy was fixed to 38%. MS/MS data was recorded in centroid mode. Isolation window was set to 0.7 m/z.

Project description:The experiment consists of M.hyp 232 cultures digests analyzed on two mass specs, LTQ Velos Pro and LTQ FT Ultra LTQ Velos Pro: Six cell culture replicates of M.hyo 232 were hydrophobically separated using tree detergents: Digitonin, Tween, and SDS. Each francion, including the insoluble pellet, was trypsin digested and then cleaned using an SCX trap follwed by a RP trap to remove detergents. Each fraction was sepated using a Dionex U3000 splitless nanoflow system operating at 333 nl per minute using a gradient of 2% ACN to 50% ACN in 4 hours. Eluate was analyzed using an LTQ Velos Pro mass spectrometer with 20 MS/MS scans of the 20 most intense peaks from each MS scan. Dynamic exclusion was enable for 3 minutes for each m/z with a repeat count of 1. LTQ FT Ultra: Two cell pellets for high resolution analysis were lysed and trypsin digested. Digested peptides were dried, resuspended in 20 mM KH2PO4, 20% ACN, pH 3 (Buffer A) in 2.5 µL and transferred to low retention vials in preparation for separation using an Ultimate 3000 configured for 2D-LC. Each sample was loaded at 15 µl/min onto an SCX microtrap for the first dimension of separation, involving SCX steps of Buffer A + 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM KCl. For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap with 2% ACN, 0.1% FA at 5 µl/min. Once desalted, the microtrap was switched into line with a fritless nano column (75µm x ~10cm) containing C18 media (5µ, 200 Å Magic, Michrom). Peptides were eluted using a gradient of 2% to 36% ACN, 0.1% FA at 350 nl/min over 60 min and electrospray ionized for analysis using an LTQ FT Ultra mass spectrometer. A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>3,000 counts) with charge states > +2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 seconds. Analysis: X!tandem searches were performed using the Mycoplasma hyopneumoniae strain 232 reference protein set from NCBI. The only difference between the searches for the LTQ Velos and LTQ FT was the precursor mass tolenance being set to +-1500ppm and +-24ppm respectively. Decoy searches were performed and the data filtered at e-value <= 0.01 with single peptide proteins discarded. These results are included in the submission as two tab-delimited text files.

Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

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