A benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics

Isobaric chemical tag labeling for quantification of intact proteins in complex samples is limited due to the tendency of intact proteins precipitate under labeling conditions and increased sample complexity as a result of side products (i.e., incomplete labeling or labeling of unintended residues). To reduce precipitation under labeling conditions, we developed a technique to remove large proteoforms that allowed for the labeling and characterization of small proteoforms (<35 kDa) using top-down proteomics. We also systematically optimized protein-level Tandem Mass Tag (TMT) labeling conditions to obtain optimal labeling parameters for complex samples. Here, we present a benchmarking protocol for protein-level TMT labeling for quantitative top-down proteomics, including complex intact protein sample preparation, protein-level TMT labeling, top-down LC/MS analysis, and TMT reporter ion quantification.• An optimized protocol for protein-level TMT labeling in complex sample.• Limits production of incorrectly labeled side products for minimization of spectral complexity.• A guideline for isobaric chemical tag quantification in top-down proteomics.


Method details
HeLa cell culture and cell lysate preparation Detailed procedure is shown in in Supplementary Document .

Procedures
The sample preparation workflow for protein-level TMT labeling is shown in Fig. 1 A.

Sample preparation prior to TMT labeling.
3   3.4. Proteins bound on trapping column are then eluted onto a C5 RPLC capillary column using a modified Thermo Scientific (Waltham, MA, USA.) Accela LC system for seperation [ 5 , 8 ]. A 200min gradient from 10% to 70% of MPB is applied for protein separation at a flow rate of 400 nL/min. The LC eluent is analyzed by an Orbitrap Exploris 240 mass spectrometer in positive mode (Thermo Fisher Scientific, Bremen, Germany) using a customized nano-ESI interface [3] .
3.4.1. Gradient time can be adjusted according to sample requirements. 3.5. Parameters used for Orbitrap Exploris 240 mass spectrometer have been previously reported [2] . Briefly, the temperature of the inlet capillary is set to 275 °C and the spray voltage is 2.   [10] deconvolution with parameters as follows: MS1 signal-to-noise ratio as 3, MS2 signal-to-noise as 1, the precursor window size as 3.0 m/z, maximum mass as 50,0 0 0 Dalton, maximum charge as 30, and m/z error as 0.02. Parameters not given here are default.
2.1.2. TopPIC [10] is utilized for identification against the annotated Human protein database (UniProt 2021-05-14, 20380 species). Parameters for TopPIC identification are decoy database searching with FDR = 0.01 for spectrum and proteoform level. The maximum number of mass shifts is 2 and the mass shift range is ± 500 Dalton. Input a text file with the PTMs given in Table 1 is used as a fixed modification file. Parameters not given here are default.

Data visualization: Protein identification can be confirmed and visualized using ProSight
Lite [11] . Load the protein mass list, precursor monoisotopic mass, and sequence obtained for the protein of interest from TopPIC into ProSight to visualize fragmentation. Appropriate PTMs or TMT tags can be manually placed on the amino acid residues to obtain the best fragmentation patterns. As shown in Fig. 3 , cyclin-dependent kinases regulatory subunit 1 (Uniprot ID: P61024) from TMT-labeled HeLa cell lysate demonstrates there is an acetylation at the N-terminal and a TMT tag on each lysine residue. Quantification result can be obtained from the relative intensity of the reporter ions.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.