Untargeted Top-down Proteomics by LC-MS/MS on Eclipse

Samples were analyzed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer in line with a Dionex Ultimate 3000 RSLCnano system

Samples (6µL ) 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 0h 10m 0s with 100% loading buffer (94.8% water:5% acetonitrile:0.2% formic acid) at 3 uL/min.

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:

A	B	C
Time (min)	%B	Valve Position
0	5	10_1
10	5	1_2
13	15
70	45
72	95
76	95
80	5
90	5

For fraction 5 and later, nanopump used the following gradient:

A	B	C
Time (min)	%B	Valve Position
0	5	10_1
10	5	1_2
13	15
70	50
72	95
76	95
80	5
90	5

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).

A	B
	High-High
Spray voltage	1600
Sweep gas	0
Ion transfer tube temp	320
Application mode	Intact Protein
Pressure mode	Low Pressure
Advanced Peak Determination	True
Default charge state	15
S-lens RF	30
Source fragmentation	15 eV

Global MS parameters

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

A	B
	High-High
Detector type	Orbitrap
Resolving power	120000
m/z RP measured	200 m/z
Scan range	600-2000
Mass range	Normal
AGC target	2000000
Normalized AGC target	500%
Max Injection Time	50 ms
Microscans	1
Data type	Profile
Polarity	Positive
Use wide quad isolation	True

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:

A	B
Intensity minimum	5000
Intensity maximum	1E20
Included charge states	4-60
Include undetermined charge states	False
Dynamic exclusion after n times	1
Dynamic exclusion duration	60 s
Mass tolerance	0.5 m/z
Exclude isotopes	True

Precursor selection filters for DDA

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

A	B
	High-High
Detector type	Orbitrap
Isolation mode	Quadrupole
Resolving power	60000
m/z RP measured	200 m/z
Scan range	350-2000
AGC target	1000000
Normalized AGC target	2000%
Max injection time	600 ms
Microscans	1
Isolation window	3 m/z
Activation type	HCD
Collision energy	32
Collision energy mode	Fixed
Polarity	Positive

Parameters for MS2 acquisition