Staggered multistep elution solid‐phase extraction capillary electrophoresis/tandem mass spectrometry: a high‐throughput approach in protein analysis

Abstract

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.

Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry for Proteomic Analysis

“…Electrokinetic elutions use EOF to drive the elution solvent through the SPME cartridge and can even allow for staggered, overlapping elutions to reduce analysis time. 31 We tested different volumes using electrokinetic elutions to maximize peptide identifications. The results of this consideration are in Figure 3A where we plotted the number of peptide identifications as a function of the elution power, the product of the electric field and the elution time (Et).…”

“…Solid-phase microextraction (SPME) has been previously shown to allow for both preconcentration of peptides and sequential elution of peptide fractions for improved proteomic coverage. 27-31 When used in conjunction with tITP, further sensitivity gains have been shown. 32, 33 In the work described here, a SPME cartridge was employed to both concentrate samples and perform sequential elution to improve comprehensiveness prior to tITP-CE-MS/MS using the high sensitivity porous sprayer.…”

Detachable Strong Cation Exchange Monolith, Integrated with Capillary Zone Electrophoresis and Coupled with pH Gradient Elution, Produces Improved Sensitivity and Numbers of Peptide Identifications during Bottom-up Analysis of Complex Proteomes

“…reported a staggered multistep elution SPE system for analysis of the tryptic digest of a set of six standard proteins. 15 Multistep elution SPE prior to CZE not only extends the separation capacity for analysis of peptide mixtures but also decreases the number of co-eluting peptide, producing an increased sequence coverage compared to single-step elution SPE-CZE/MS/MS. Wang et al described a C8 SPE, multistep elution, tITP CZE-MS/MS procedure for the proteomic analysis of a Pyrococcus furiosus tryptic digest.…”

A Miniaturized Transverse Flow Gating Interface for the On-line Coupling of Solid-phase Extraction with Capillary Electrophoresis

“…In addition to implementation of electric connection for CE, the interface should have the ability to exactly transfer nanoliters of fractions from SPE onto CE with minimized degradation of the efficiencies of SPE and CE. For such a purpose, a variety of interfaces have been designed, including such types as valve-loop, 7,8 T-split, 9,10 valve-loop-vial or T-piece, [11][12][13][14] two-leveled two cross, 15,16 flow-gating, 11,[17][18][19][20][21] and interfaces with integrated SPE. 22,23 With the first three types of interfaces, success of the coupling has been achieved to some extent, but some limitations have been recognized.…”

“…However, the position of the SPE outlet relative to the CE capillary is critical to the performance of the SPE-CE system, and contamination of the CE capillary from the SPE effluent has been observed. 15,16 For the interface with integrated SPE, as the SPE column is part of the CE system, some deleterious influences of the SPE sorbent may occur on the CE system during analysis. 22 Most of the aforementioned interfaces require relatively complicated switching procedures that may result in poor reproducibilities.…”