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

Abstract

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.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry

“…Spectral simplification can counterbalance these effects. Recent studies have demonstrated that the use of field asymmetric ion mobility spectrometry (FAIMS) can lead to deeper characterization of intact proteomes in the 0–30 kDa mass range. − Three recent studies by Tholey and co-workers have first described the benefits of internal stepping of the compensatory DC voltages (compensation voltage, C.V.) applied to the central electrode of the FAIMS apparatus during a single LC–FAIMS–MS 2 analysis of proteoforms from CaCo-2 cells, , and then proven the compatibility of such data acquisition method with off-line protein fractionation via passively eluting proteins from polyacrylamide gels as intact species (PEPPI) . In the latter study, FAIMS-enabled gas-phase fractionation of intact protein ions led to the identification of more than 8500 low-molecular weight (MW) proteoforms.…”

Multidimensional separations in top–down proteomics

“…57 As shown in for MD separation and TDP. 59 In summary, good orthogonality, improved separation efficiency, and more comprehensive protein characterisation has been achieved using high-pH/low-pH RPLC platforms compared with 1D RPLC for TDP.…”

“…This group utilised low‐pH RPLC in both dimensions and employed different ion pairing reagents and stationary phases to change selectivity between the two RPLC dimensions. In addition to concatenation to improve sample throughput, this group demonstrated the first implementation of low‐pH/low‐pH RPLC for MD separation and TDP 59 …”

Improved label-free quantification of intact proteoforms using field asymmetric ion mobility spectrometry

“…Recent studies have demonstrated that the use of field asymmetric ion mobility spectrometry (FAIMS) 22 can lead to a deeper characterization of intact proteomes in the 0-30 kDa mass range. [23][24][25] Three recent studies by Tholey and co-workers have first described the benefits of internal stepping of the compensatory DC voltages (compensation voltage, C.V.) applied to the central electrode of the FAIMS apparatus during a single LC-FAIMS-MS 2 analysis of proteoforms from CaCo-2 cells, 26,27 and then proven the compatibility of such data acquisition method with off-line protein fractionation 28 via passively eluting proteins from polyacrylamide gels as intact species (PEPPI). 29 In the latter study, FAIMS-enabled gas-phase fractionation of intact protein ions led to the identification of more than 8,500 low-molecular weight (MW) proteoforms.…”