Feb 28, 2022

Public workspaceUntargeted Top-down Proteomics by LC-MS/MS on Eclipse

DOI
dx.doi.org/10.17504/protocols.io.bttknnkw
  • 1Purdue University;
  • 2Northwestern University
Kelleher KRG KRG Research Group
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DOI: dx.doi.org/10.17504/protocols.io.bttknnkw
Protocol Citation: Bryon Drown, Jeannie Camarillo, Rafael Melani, Neil Kelleher 2022. Untargeted Top-down Proteomics by LC-MS/MS on Eclipse. protocols.io https://dx.doi.org/10.17504/protocols.io.bttknnkw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 30, 2021
Last Modified: February 28, 2022
Protocol Integer ID: 48716
Funders Acknowledgement:
Office of the Director of the National Institutes of Health
Grant ID: UH3 CA24663
National Institute of General Medical Sciences of the National Institutes of Health
Grant ID: P41 GM108569
National Institute of Cancer of the National Institute of Cancer of the National Institutes of Health
Grant ID: F32 CA246894
Abstract
Describes the LC-MS/MS data acquisition procedure for top-down proteomics samples using the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer
Materials
PLRP-S 5-μm particles 1,000-Å pore size (Agilent Technologies)
Water Optima LC/MS Grade (Fisher Scientific #W64)
Acetonitrile Optima LC/MS Grade (Fisher Scientific #A955-4)
Formic Acid LC/MS Grade (Thermo Scientific #28905)
15 μm SilicaTip PicoTip Emitter (New Object #FS360-50-15-N-20-C12)

Buffer A: 94.8 % water, 5 % acetonitrile, 0.2 % formic acid
Buffer B: 4.8 % water, 95 % acetonitrile, 0.2 % formic acid
Samples were analyzed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer in line with a Dionex Ultimate 3000 RSLCnano system
Samples (Amount6 µL ) were injected via the autosampler and loaded onto a self-packed trap column (150 μm i.d. x 2 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) for Duration00:10:00 with 100% loading buffer (94.8% water:5% acetonitrile:0.2% formic acid) at 3 uL/min.

10m
Following a valve switch and initiation of the nanopump at 300 nL/min (buffer A: 94.8 % water, 5 % acetonitrile, 0.2 % formic acid; buffer B: 4.8 % water, 95 % acetonitrile, 0.2 % formic acid), proteins were separated on a self-packed analytical column (75 μm i.d. x 25 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) according to the following gradient for fractions 1-4:
ABC
Time (min)%BValve Position
  0  510_1
10  51_2
1315
7045
7295
7695
80  5
90  5
For fraction 5 and later, nanopump used the following gradient:

ABC
Time (min)%BValve Position
0510_1
1051_2
1315
7050
7295
7695
805
905

Eluted proteins were ionized in positive ion mode nanoelectrospray ionization (nESI) using a pulled tip nanospray emitter (15-μm i.d. ×125 mm) packed with 1mm of PLRP-S 5-μm particles 1,000-Å pore size with a custom nano-source (https://proteomicsresource.washington.edu/docs/protocols05/UWPR_NSI_Source.pdf).


AB
High-High
Spray voltage1600
Sweep gas0
Ion transfer tube temp320
Application modeIntact Protein
Pressure modeLow Pressure
Advanced Peak DeterminationTrue
Default charge state15
S-lens RF30
Source fragmentation15 eV
Global MS parameters

Precursor (intact protein) spectra were acquired at 120k FTRP.

AB
High-High
Detector typeOrbitrap
Resolving power120000
m/z RP measured200 m/z
Scan range600-2000
Mass rangeNormal
AGC target2000000
Normalized AGC target500%
Max Injection Time50 ms
Microscans1
Data typeProfile
PolarityPositive
Use wide quad isolationTrue
Parameters for MS1 acquisition

The mass spectrometer was operated using a TopN 3 sec data-dependent acquisition mode
Precursor ions were filtered by intensity, charge state, and dynamic exclusion:

AB
Intensity minimum5000
Intensity maximum1E20
Included charge states4-60
Include undetermined charge statesFalse
Dynamic exclusion after n times1
Dynamic exclusion duration60 s
Mass tolerance0.5 m/z
Exclude isotopesTrue
Precursor selection filters for DDA

Ions for fragmentation were isolated and fragmented via higher energy dissociation (HCD):

AB
High-High
Detector typeOrbitrap
Isolation modeQuadrupole
Resolving power60000
m/z RP measured200 m/z
Scan range350-2000
AGC target1000000
Normalized AGC target2000%
Max injection time600 ms
Microscans1
Isolation window3 m/z
Activation typeHCD
Collision energy32
Collision energy modeFixed
PolarityPositive
Parameters for MS2 acquisition