Identification of proteoforms by top‐down proteomics using two‐dimensional low/low pH reversed‐phase liquid chromatography‐mass spectrometry

In top‐down (TD) proteomics, efficient proteoform separation is crucial to reduce the sample complexity and increase the depth of the analysis. Here, we developed a two‐dimensional low pH/low pH reversed‐phase liquid chromatography separation scheme for TD proteomics. The first dimension for offline...

Full description

Saved in:
Bibliographic Details
Published in:Proteomics (Weinheim) 2024-02, Vol.24 (3-4), p.e2200542-n/a
Main Authors: Kaulich, Philipp T., Cassidy, Liam, Tholey, Andreas
Format: Article
Language:English
Subjects:
2D‐LC
Ammonium
Chromatography
FAIMS
Formic acid
Fractionation
High temperature
Ionic mobility
Liquid chromatography
Mass spectrometry
Mass spectroscopy
offline fractionation
pH effects
proteoform separation
Proteomics
Reagents
Scientific imaging
Separation
Time measurement
top‐down proteomics
Trifluoroacetic acid
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In top‐down (TD) proteomics, efficient proteoform separation is crucial to reduce the sample complexity and increase the depth of the analysis. Here, we developed a two‐dimensional low pH/low pH reversed‐phase liquid chromatography separation scheme for TD proteomics. The first dimension for offline fractionation was performed using a polymeric reversed‐phase (PLRP‐S) column with trifluoroacetic acid as ion‐pairing reagent. The second dimension, a C4 nanocolumn with formic acid as ion‐pairing reagent, was coupled online with a high‐field asymmetric ion mobility spectrometry (FAIMS) Orbitrap Tribrid mass spectrometer. For both dimensions several parameters were optimized, such as the adaption of the LC gradients in the second dimension according to the elution time (i.e., fraction number) in the first dimension. Avoidance of elevated temperatures and prolonged exposure to acidic conditions minimized cleavage of acid labile aspartate–proline peptide bonds. Furthermore, a concatenation strategy was developed to reduce the total measurement time. We compared our low/low pH with a previously published high pH (C4, ammonium formate)/low pH strategy and found that both separation strategies led to complementary proteoform identifications, mainly below 20 kDa, with a higher number of proteoforms identified by the low/low pH separation. With the optimized separation scheme, more than 4900 proteoforms from 1250 protein groups were identified in Caco‐2 cells.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.202200542