Improved Sensitivity and Separations for Phosphopeptides using Online Liquid Chromotography Coupled with Structures for Lossless Ion Manipulations Ion Mobility-Mass Spectrometry

Abstract

Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, quantification, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integrating structures for lossless ion manipulations ion mobility-mass spectrometry (SLIM IM-MS) with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges. A mixture of 51 heavy-labeled phosphopeptides was analyzed with a SLIM IM module having integrated ion accumulation and long-path separation regions. The SLIM IM-MS provided limits of detection as low as 50-100 pM (50-100 amol/μL) for several phosphopeptides, with the potential for significant further improvements. In addition, conventionally problematic phosphopeptide isomers could be resolved following an 18 m SLIM IM separation. The 2-D LC-IM peak capacity was estimated as ∼9000 for a 90 min LC separation coupled to an 18 m SLIM IM separation, considerably higher than LC alone and providing a basis for both improved identification and quantification, with additional gains projected with the future use of longer path SLIM IM separations. Thus, LC-SLIM IM-MS offers great potential for improving the sensitivity, separation, and throughput of phosphoproteomics analyses.

Figure 1.

Arrival time distributions for ESI of a 25 nM target peptide solution showing: (A) TELISpVSEVHPSR²⁺ (m/z 772.373) with SLIM IM module accumulation times of 1, 2, 2.5, 3, and 5 s, and (B) YLSFTpPPEKDGFPSGTPALNAK²⁺ (m/z 1213.082) with SLIM IM module accumulation times of 1, 2, and 3 s, demonstrating increased S/N.

Figure 2.

Arrival time distributions for target phosphopeptide isomers TPSpSEEISPTK²⁺ and TPSSEEISpPTK²⁺ after (A) 4.5 and (B) 18 m of separation, demonstrating improved resolution for longer path SLIM IM separations.

Figure 3.

Overlay of selected ion chromatograms for the 51 targeted heavy-labeled phosphopeptides in a S. oneidensis proteome digest matrix.

Figure 4.

Extracted ion chromatograms for target LGTGFNPNTp-LDKQK²⁺ (m/z 810.897), which elutes at 37.2 min, both (A) without SLIM IM-filtering and (B) with SLIM IM filtering at 322 ms.

Figure 5.

(A) Log/log plot of IM filtered intensity vs concentration for target peptide GSDASpGQLFHGR²⁺ (m/z 661.282), demonstrating approximately 4 orders of linear dynamic range. The improvement in S/N is primarily a result of IM filtering, in which a coeluting interference at very similar m/z is filtered out from the target peak (arrival time 246 ms) at (B) 1 nM and (C) 50 pM.

Figure 6.

Arrival time distributions following 18 m SLIM IM separation for 5 nM concentrations of the target singly phosphorylated peptide isomer pairs: (A) TPS(p)SEEIS(p)PTKFPGLYR²⁺ (m/z 999.976) and (B) TPS(p)SEEIS(p)PTK²⁺ (m/z 632.283), where the two phosphoserine residue sites are indicated by red.