Samples were reconstituted in 67.5 µL of 0.1% formic acid. An aliquot of 4.5 μL was analysed on an ETD enabled Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, US) connected to an EASY-nLC system (Proxeon Biosystem, West Palm Beach, FL, US) through a Proxeon nanoelectrospray ion source. Peptides were separated by a 2–90% acetonitrile gradient in 0.1% formic acid using a PicoFrit analytical column (20 cm × ID75 μm, 5-μm particle size, New Objective) at a flow rate of 300 nL min−1 for 85 minutes. The nanoelectrospray voltage was set to 2.2 kV, and the source temperature was 275 °C. All instrumental methods were set up in the data-dependent acquisition mode (DDA). Full scan MS spectra (300–1600 m/z) were acquired in the Orbitrap analyser after accumulation to a target value of 1 × 106. The resolution in the Orbitrap was set to r = 60.000, and the 20 most intense peptide ions with charge state ≥2 were sequentially isolated to a target value of 5.000 and fragmented in the linear ion trap using low-energy CID (normalised collision energy of 35%). The signal threshold for triggering an MS/MS event was set to 1.000 counts. Dynamic exclusion was enabled with an exclusion size list of 500, an exclusion duration of 60 s, and a repeat count of 1. An activation of q = 0.25 and an activation time of 10 ms were used71 (link).
Picofrit analytical column
Manufactured by New Objective
Sourced in United States
The PicoFrit analytical column is a lab equipment product designed for analytical applications. It provides a platform for separation and analysis of chemical compounds. The core function of the PicoFrit column is to facilitate chromatographic separation processes.
Automatically generated - may contain errors
Lab products found in correlation
Zorbax 300sb c18 trap column, by Agilent Technologies (6 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (6 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (4 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Nanolc ultra 1d plus system, by Thermo Fisher Scientific (4 mentions)
Reprosil pur 120 c18 aq media, by Dr. Maisch (3 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (3 mentions)
Ltq orbitrap elite, by Thermo Fisher Scientific (3 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap elite mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Nanolc ultra 1d plus system, by AB Sciex (2 mentions)
Fusion lumos mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Reprosil pur media, by Dr. Maisch (2 mentions)
Orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (1 mentions)
Betabasic 5 μm 300 particles, by New Objective (1 mentions)
Formic acid, by Merck Group (1 mentions)
19 protocols using picofrit analytical column
1
Proteomic Analysis of Peptides by LC-MS/MS
2
Identifying PDZ Domain Interacting Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify novel proteins bound to the PDZ domain of SNX27, samples were electrophoresed on SDS-polyacrylamide gels, and protein bands were visualized by staining with Coomassie blue. The band of interest was excised and digested with trypsin at 1:50 enzyme/substrate ratio. The dried tryptic digest was analyzed on an LTQ-Orbitrap XL (Thermo-Fisher Scientific LLC) interfaced with an Eksigent nano-LC 1D plus system (Eksigent Technologies LLC, Dublin, CA) using CID fragmentation. Samples were loaded onto an Agilent Zorbax 300SB-C18 trap column at a flow rate of 5 µl/min for 10 min, and then separated on a reversed-phase PicoFrit analytical column (New Objective, Woburn, MA) using a 40-min linear gradient of 2–40% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. LTQ-Orbitrap XL settings were as follows: spray voltage 1.5 kV; full MS mass range: m/z 200 to 2000. The LTQ-Orbitrap XL was operated in a data-dependent mode; i.e. MS1 in the ion trap, scan for precursor ions followed by six data-dependent MS2 scans for precursor ions above a threshold ion count of 2000 with collision energy of 35%. The raw file generated from the LTQ-Orbitrap XL was analyzed as described [11 (link)].
3
Peptide Separation and Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptides obtained from co-immunoprecipitations were separated on a 25 cm, 75 μm internal diameter PicoFrit analytical column (New Objective) packed with 1.9 μm ReproSil-Pur 120 C18-AQ media (Dr. Maisch) using an EASY-nLC 1000 or EASY-nLC 1200 (Thermo Fisher Scientific). The column was maintained at 50°C. Buffer A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile or 0.1% formic acid in 80% acetonitrile. Peptides were separated on a segmented gradient from 6% to 25% or 31% buffer B. Eluting peptides were analyzed on an Orbitrap Fusion or QExactive HF mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurements were carried out at 60000 resolution in the 300 to 1500 or 1800 m/z range. The ten most intense precursors with charge state from two to seven only were selected for HCD fragmentation using 27% or 25% normalized collision energy. The m/z values of the peptide fragments were measured in the orbitrap at a resolution of 30000 using an AGC target of 2e5 and 55, 80 or 100 ms maximum injection time. Upon fragmentation, precursors were put on a dynamic exclusion list for 45 s.
4
Peptide Analysis by Orbitrap Velos Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peptide mixture (4.5 µL) was analyzed using an LTQ Orbitrap Velos (Thermo Fisher Scientific) mass spectrometer coupled to nanoflow liquid chromatography on an EASY-nLC system (Proxeon Biosystems) with a Proxeon nanoelectrospray ion source. Peptides were subsequently separated in a 2–90% acetonitrile gradient in 0.1% formic acid using a PicoFrit analytical column (20 cm × ID75, 5 µm particle size, New Objective) at a flow rate of 300 nL/min over 212 min, in which a gradient of 35% acetonitrile is reached in 175 min. The nanoelectrospray voltage was set to 2.2 kV, and the source temperature was set to 275 °C. The instrument methods employed for LTQ Orbitrap Velos were set up in DDA mode. Full scan MS spectra (m/z 300–1600) were acquired in the Orbitrap analyzer after accumulation to a target value of 1e6. Resolution in the Orbitrap was set to r = 60,000, and the 20 most intense peptide ions (top 20) with charge states ≥2 were sequentially isolated to a target value of 5000 and fragmented in the high-pressure linear ion trap by CID (collision-induced dissociation) with a normalized collision energy of 35%. Dynamic exclusion was enabled with an exclusion size list of 500 peptides, an exclusion duration of 60 s and a repetition count of 1. An activation Q of 0.25 and an activation time of 10 ms were used. The run order of the samples is described in Supplementary Data 2 .
5
Peptide Separation and Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peptides were separated on a 25 cm, 75 μm internal diameter PicoFrit analytical column (New Objective) packed with 1.9 μm ReproSil-Pur 120 C18-AQ media (Dr. Maisch) using an EASY-nLC 1200 (Thermo Fisher Scientific). The column was maintained at 50°C. Buffer A and B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile. Peptides were separated on a segmented gradient from 6% to 31% buffer B for 45 min and from 31% to 50% buffer B for 5 min at 200 nl/min. Eluting peptides were analyzed on a QExactive HF mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurements were carried out at 60,000 resolution in the 300 to 1800 m/z range. The top ten most intense precursors with charge state from 2 to 7 only were selected for HCD fragmentation using 25% normalized collision energy. The m/z values of the peptide fragments were measured at a resolution of 30,000 using a minimum AGC target of 8e3 and 55 ms maximum injection time. Upon fragmentation, precursors were put on a dynamic exclusion list for 45 sec.
6
Quantitative Proteomics by LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC-MS/MS was performed using an Eksigent nanoLC-Ultra 1D plus system (Dublin, CA) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher) using CID fragmentation. Peptides were first loaded onto a Zorbax 300SB-C18 trap column (Agilent, Santa Clara, CA) at a flow rate of 6 μL/minute for 6 minutes, and then separated on a reversed-phase PicoFrit analytical column (New Objective, Woburn, MA) using a 40-minute linear gradient of 5–40% acetonitrile in 0.1% formic acid at a flow rate of 250 nL/minute. LTQ Orbitrap Elite settings were as follows: spray voltage 1.5 kV, and full MS mass range: m/z 230 to 2000. The LTQ Orbitrap Elite was operated in a data-dependent mode (i.e., one MS1 high resolution [60,000] scan for precursor ions followed by six data-dependent MS/MS scans for precursor ions above a threshold ion count of 2000 with collision energy of 35%). Raw files generated from the LTQ Orbitrap Elite were analyzed using Proteome Discoverer 1.4 (Thermo Fisher) with the MASCOT database search engine. The following search criteria were used: database, Swiss-Prot (Swiss Institute of Bioinformatics); taxonomy, Mus musculus (mouse); enzyme, trypsin; miscleavages, 3; variable modifications, Oxidation (M), Nethylmaleimide (C), Deamidation (NQ); MS peptide tolerance, 25 ppm; MS/MS tolerance, 0.8 Da. Peptides were filtered at a false discovery rate (FDR) of 1%.
7
Mass Spectrometry Proteomics Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled with an Eksigent NanoLC-Ultra 1D Plus system. Peptides were loaded onto a Zorbax 300SB-C18 trap column (Agilent) at a flow rate of 6 μL/min for 6 minutes and then separated on a reversed-phase PicoFrit analytical column (New Objective) using a short 15-minute linear gradient of 5% to 40% acetonitrile in 0.1% formic acid at a flow rate of 250 nL/min for 2D-DIGE protein spots and a 40-minute linear gradient for SNO-RAC samples. Mass analysis was carried out in data-dependent analysis mode, in which MS initially scanned the full MS mass range from m/z 300 to 2000 at 60 000 mass resolution, and then 6 collision-induced dissociation MS scans were sequentially carried out in the Orbitrap and the ion trap, respectively.
8
Peptide Identification by LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An aliquot of 4.5 uL of the resulting mixture of peptides of each sample was loaded on a mass spectrometer LTQ Orbitrap Velos (Thermo Fisher Scientific, Waltham, MA, USA) connected to a nanoflow LC (LC-MS/MS) instrument by an EASY-NLC system (Proxeon Biosystems, West Palm Beach, FL, USA) with a Proxeon nanoelectrospray ion source. Peptides were separated by 2%-90% acetonitrile gradient in 0.1% formic acid using a PicoFrit analytical column (20 cm x ID75 μm, 5 μm particle size, New Objective, Woburn, MA, USA), with a flow rate of 300 nL/min for 45 minutes. The nanoelectrospray voltage was set to 1.7 kV and the source temperature was 275°C. All the equipment instrumental methods were set up in the data-dependent acquisition mode. Full scan of MS spectra (m/z 300–1600) were acquired by the Orbitrap analyzer after accumulation to a target value of 1e6. The resolution was set to the r = 60,000 and the 5 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 5,000 and fragmented in the linear ion trap using low-energy CID (normalized collision energy of 35%). The signal threshold to trigger an MS/MS event was set to 500 counts. Dynamic exclusion was enabled with an exclusion size list of 500, exclusion duration of 60 seconds, and repeat count of 1. An activation q = 0.25 and activation time of 10 ms were used [21 (link)].
9
Mass Spectrometry Analysis of Microdissected Peptides
10
Peptide Identification via Orbitrap LC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An aliquot containing 4.5µL of peptide mixture was analyzed on an LTQ Orbitrap Velos (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer coupled to nanoflow liquid chromatography on an EASY-nLC system (Proxeon Biosystems, Odense, Dinamarca) through a Proxeon nanoelectrospray ion source. Peptides in 0.1% formic acid were separated using a 2–90% acetonitrile gradient in a PicoFrit analytical column (20 cm × ID75, 5 µm particle size, New Objective) with a flow rate of 300 nL/min over 212 min and a gradient of 35% acetonitrile at 175 min. The nanoelectrospray voltage was set to 2.2 kV and source temperature to 275°C. Instrument methods for the LTQ Orbitrap Velos were set up in the data-dependent acquisition mode. Full scan MS spectra (m/z 300–1600) were acquired in the Orbitrap analyzer after accumulation to a target value of 1e6. The resolution in the Orbitrap was set to r = 60,000, and the 20 most intense peptide ions with charge states ≥ 2 were sequentially isolated to a target value of 5000 and fragmented in the high-pressure linear ion trap through CID (collision-induced dissociation) with a normalized collision energy of 35%. Dynamic exclusion was enabled with an exclusion size list of 500 peptides, exclusion duration of 60 s duration, and repetition count of 1. An activation Q of 0.25 and activation time of 10 ms were used.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!