Multi-Segment Direct Inject nano-ESI-LTQ-FT-ICR-MS/MS For Protein Identification

Abstract

Reversed phase high performance liquid chromatography (HPLC) interfaced to electrospray tandem mass spectrometry (MS/MS) is commonly used for the identification of peptides from proteolytically cleaved proteins embedded in a polyacrylamide gel matrix as well as for metabolomics screening. HPLC separations are time consuming (30-60 min average), costly (columns and mobile phase reagents), and carry the risk of column carry over between samples. The use of a chip-based nano-ESI platform (Advion NanoMate) based on replaceable nano-tips for sample introduction eliminates sample cross-contamination, provides unchanging sample matrix, and enhances spray stability with attendant increases in reproducibility. Recent papers have established direct infusion nano-ESI-MS/MS utilizing the NanoMate for protein identification of gel spots based on full range MS scans with data dependent MS/MS. In a full range scan, discontinuous ion suppression due to sample matrix can impair identification of putative mass features of interest in both the proteomic and metabolomic workflows. In the current study, an extension of an established direct inject nano-ESI-MS/MS method is described that utilizes the mass filtering capability of an ion-trap for ion packet separation into four narrow mass ranges (50 amu overlap) with segment specific dynamic data dependent peak inclusion for MS/MS fragmentation (total acquisition time of 3 minutes). Comparison of this method with a more traditional nanoLC-MS/MS based protocol utilizing solvent/sample stream splitting to achieve nanoflow demonstrated comparable results for protein identification from polyacrylamide gel matrices. The advantages of this method include full automation, lack of cross-contamination, low cost, and high throughput.

Rapid cyclic ion mobility separations of monosaccharide building blocks as a first step toward a high-throughput reaction screening platform for carbohydrate syntheses

“…3, where our overarching goal is to couple high-resolution cIMS-MS separations with existing automated ion introduction platforms (e.g., desorption electrospray ionization, DESI, or the commercialized electrospray NanoMate by Advion). 31,[44][45][46][47] From an analytical perspective, our present results demonstrate that only 1 minute is necessary for each monosaccharide building block anomer separation and oligosaccharide isomer separation which is in good timescale agreement with the aforementioned DESI/NanoMate ionization sources (Fig. 5).…”

Quantitative shotgun proteome analysis by direct infusion

“…Several papers describe qualitative analysis of peptides from simple mixtures by direct infusion, an approach that is already common approach in metabolomics 6,7 . Twenty-five years ago, direct infusion of peptides from trypsin-digested gel bands or standard proteins was common, though offered limited depth, typically less than 60 peptides [8][9][10][11][12][13] . As LC and MS co-evolved, LC-MS became the premiere technology for the analysis of the tremendously complex mixture of peptides that results from whole proteome digestion.…”

Identification of two arginine kinase forms of endoparasitoid Leptomastix dactylopii venom by bottom up‐sequence tag approach

“…Moreover, HPLC‐MS/MS is not easily practicable when a large‐scale sample concentration is not a flexible option, as in the case of parasitoid wasp venoms . The use of electrospray ionization (ESI) direct infusion eliminates sample cross‐contamination, increases the reproducibility and speeds time of analysis . Here, we created a custom‐made transcriptomic database from the L .…”

“…[23] The use of electrospray ionization (ESI) direct infusion eliminates sample cross-contamination, increases the reproducibility and speeds time of analysis. [24] Here, we created a custom-made transcriptomic database from the L. dactylopii venom glands by applying the high-throughput RNA sequencing (RNA-Seq) approach. Moreover, the venom components were separated by twodimensional gel electrophoresis (2DE), and trypsinized 2DE protein spots were analyzed by a direct inject Electrospray Ionization double Quadrupole-Fourier Transformation-Ion Cyclotron Resonance/ Tandem mass spectrometry (ESI qQ-FT-ICR-MS/MS) method within few minutes.…”