Thaw peptide samples On ice , and transfer 30µLof each sample to LC autosampler vials (Agilent, Cat.#5182-0567,#5182-0564) or 96-well plate (Bio-Rad, Cat.#HSP9655).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is performed with a Thermo Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA).

Equipment

Value	Label
Obitrap Exploris 480	NAME
Mass spectrometer	TYPE
Thermo Fisher	BRAND
BRE725532	SKU

Equipment

Value	Label
1290 Infinity UHPLC	NAME
Ultra-high performance liquid chromatography system	TYPE
Agilent Technologies	BRAND
1290 Infinity UHPLC	SKU

Samples were loaded into a temperature controlled autosampler operating at 4°C. The separation on the UHPLC is achieved by using an Agilent InfinityLab Poroshell 120 EC-C18 (2.1 mm ID,100 mm length,1.9 µm particle size, 120-Å pore size) (Agilent, Cat.#695675-902) coupled with an Agilent InfinityLab Poroshell 120 EC-C18 guard column (2.1 mm ID,5 mm length,1.9 µm particle size, 160-Å pore size)(Agilent, Cat.#821725-940). The column is operated at 60°C.

Twenty micrograms 20µg of peptides are loaded onto the column from each sample and separated using a gradient separation with 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) operating at a flow rate of 0.4 ml/min. A 15 minute total acquisition time with a 10 minute linear elution gradient of chromatographic separation is as follows:

A	B	C	D
Step	%A	%B	Time (minute)
1	98	2	0.0
2	98	10	1.5
3	65	35	11.5
4	20	80	12.0
5	20	80	13.5
6	98	2	14.0
7	98	2	15.0

Table 1. Chromatographic gradient table

The eluted peptides were ionized via OptaMax^TM NG Electrospray Ion Source operating in positive ion mode with the following source parameters:

A	B
Vaporizer temp	250 °C
Ion transfer tube temp	300 °C
Positive ion voltage	3500 V
Shealth gas	50
Aux gas	20

Table 2. Source conditions

The mass spectrometer is operated in data independent mode with a duty cycle of 3 survey scans and 45 MS2 scans . The survey scan and MS2 scan parameters are as follows:

A	B
Survey scan obitrap resolution	60K
Survey scan MS range	380 to 985 m/z
Survey scan AGC target	300%
Survey scan maximum ion injection time	45 ms
DIA precusor isolation window	13.5 m/z
MS2 scan range	145 to 1450 m/z
MS2 scan obitrap resolution	15K
MS2 scan AGC target	1000%
MS2 scan maximum ion injection time	22 ms

DIA parameters

The MS raw data were acquired using Thermo Scientific Xcalibur version 4.3.73

Software

Value	Label
Thermo Fisher Scientific	NAME
Thermo Fisher Scientific	DEVELOPER
http://www.thermofisher.com/order/catalog/product/OPTON-30965#/OPTON-30965	LINK
4.3.73	VERSION

The acquired DIA raw data files were analyzed by an integrated software suite DIA-NN v 1.8.1.

DIA-NN configurations for Library-free search and peptide quantification:

A	B
Enzyme	Trypsin
Maximum missed cleavages	1
Precusor and Fragment MS accuricies	Automatically determined
Precusor length range	7-30
Precusor charge range	2-4
Fixed modifications	Carbamidomethyl (Cys)
Variable modifications	Deamination (Asn, Gln); Oxidation (Met)
Precusor and protein identificaiton FDR	1%
Quantification strategy	Robust LC
Spectral library	Generated from fasta files of latest proteomes at Uniprot

DIA-NN configurations

Protein quantity reported by DIA-NN was further processed and visualized using an jupyter notebook described in detail through an established protocol.

Label-free quantification (LFQ) proteomic data analysis from DIA-NN output files

The Exploris mass spectrometer is subjected to mass calibration check prior to analyzing samples to verify mass accuracy, intensity, and resolution of ions using Pierce™ FlexMix™ Calibration Solution purchased from Thermo Fisher Scientific.

A weekly mass calibration is performed to maintain <3 ppm mass accuracy without correction from internal calibrant.

The mass spectrometer is subjected to a system calibration at least quarterly (and more frequently, if transmission tune fails, or performance issues arise).

UHPLC-Obitrap system performance is monitored at the beginning, middle, and end of large sample sets by running full LC-MS/MS data collection of 20 ug E.coli cell lysate protein tryptic digest. The protein identification, mass accuracy, peak shape, and resolution of peptides are evaluated.