Filter-Aided Sample Prep (FASP) was used to lyse cells, digest with trypsin, and recover the tryptic peptides [9 (link)]. Peptides were analyzed by nano-LC/MS/MS. Peptides were eluted across a 3-hour gradient on a 20 cm, self-packed C18 capillary column on an Eksigent NanoLC-2D system. Mass spectra were acquired on a Thermo LTQ Velos linear ion-trap mass spectrometer. The 5 highest intensity 2+ or 3+ ions from each MS scan were selected for collision induced dissociation (CID).
Nanolc 2d system
Manufactured by AB Sciex
The NanoLC-2D system is a liquid chromatography instrument designed for high-performance separation and analysis of complex samples. It features two-dimensional separation capability, enabling advanced chromatographic techniques for enhanced analyte resolution and identification.
Automatically generated - may contain errors
Lab products found in correlation
Ltq velos, by Thermo Fisher Scientific (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
12t solarix ft icr ms, by Bruker (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Microtof time of flight mass spectrometer, by Bruker (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
6 protocols using nanolc 2d system
1
Filter-Aided Tryptic Peptide Preparation
2
LC-MS/MS Peptide Separation and Analysis
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Peptides were dissolved in solvent A (2% FA in 3% ACN) and directly loaded onto a reverse-phase Trap column (Chrom XP C18-CL-3 m 120A, Eksigent). Peptide separation was performed using a reverse-phase analytical column (3C18-CL-120, 3 µm, 120A, Eksigent) with a linear gradient of 4–22% solvent B (0.1% FA in 98% ACN) for 50 min, 22–35% solvent B for 12 min, increasing to 80% solvent B in 4 min, and then holding at 80% solvent B for the last 4 min, all at a constant flow rate of 300 mL/min on an Eksigent NanoLC 2D system. The resulting peptides were analyzed with a Triple-TOF 5600+mass spectrometer (AB Sciex).
3
Nano-LC-micro-ESI MS/MS Analysis
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Samples were analyzed on a Bruker Solarix 12T FT-ICR MS by nanoLC-micro-ESI MS/MS at a flow rate of 400 nL/min. Each sample was on-line separated with an Eksigent nanoLC 2D system over an in-house packed 75 micron i.d. nano-LC column packed with 8 cm of Phenyl hexal resin; 5 µL of sample was injected onto the column. The sample was loaded onto the column and washed for 5 min with 20%/80% A/B solvent. The sample was eluted with a gradient starting at 20%/80% A/B solvent and ramping to 1%/99% A/B solvent over 10 min. 1%/99% A/B solvent was held for 5 min to elute everything from the column. The solvent was then stepped down immediately to 20%/80% A/B solvent and held there for 10 min to re-equilibrate the column for the next sample. The solvent compositions were: Solvent A: 98% H2O, 2% MeOH, with 10 mM NH4OAc; Solvent B: 98% MeOH, 2% H2O with 10 mM NH4OAc)22 (link).
4
Proteomics Analysis by NanoLC-MS/MS
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Samples were loaded on a homemade C18 precolumn directly connected to the switching valve of a homemade reversed-phase column with a gradient from 10% to 60% acetonitrile (0.2% formic acid [FA]) and a 600 nL/min flow rate on a NanoLC-2D system (Eksigent, Dublin, CA) connected to an Q-Exactive Plus (Thermo Fisher Scientific). Each full mass spectrometry spectrum acquired with a 70,000 resolution was followed by 12 MS/MS spectra, in which the 12 most abundant multiply charged ions were selected for MS/MS sequencing.
5
Synthesis and Analysis of Ag29(LA)12 Clusters
6
Protein Identification via Mass Spectrometry
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Candidate electrophoretic protein bands were excised from acrylamide gels and sent to the Montreal Clinical Research Institute (IRCM) for sequencing. At IRCM proteins were hydrolyzed using trypsin; resulting peptides were analyzed with a nanoLC-2D system (Eksigent) and coupled to the LTQ Orbitrap Velos (ThermoFisher Scientific).
The sequences of the obtained peptides were compared with the NCBI bacterial protein database using the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi; [26] (link)). Comparisons between other bacterial amylases were made using BioEdit Sequence Alignment Editor software [27] . For prediction of protein localization and motif identification, PSORTb v.3.0 (http://psort.org/; [28] ), the Conserved Domain database (https://www.ncbi.nlm. nih.gov/cdd/; [29] (link)), and the Pfam search engine version cdd.v.3.18 sequence database (http://pfam.xfam.org/; [30] (link)) with the preset options were used.
The sequences of the obtained peptides were compared with the NCBI bacterial protein database using the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi; [26] (link)). Comparisons between other bacterial amylases were made using BioEdit Sequence Alignment Editor software [27] . For prediction of protein localization and motif identification, PSORTb v.3.0 (http://psort.org/; [28] ), the Conserved Domain database (https://www.ncbi.nlm. nih.gov/cdd/; [29] (link)), and the Pfam search engine version cdd.v.3.18 sequence database (http://pfam.xfam.org/; [30] (link)) with the preset options were used.
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