Development and Evaluation of Atmospheric-Pressure Electron Capture Dissociation (AP-ECD) for the LC/MS Analysis of Protein Digests

Damon Robb; Davin Carter; Jason Rogalski ; Juergen Kast ; Michael Blades

University of British Columbia, Vancouver, CANADA

Novel Aspect

update on the development and performance of a novel AP-ECD source

Introduction

Atmospheric-pressure electron capture dissociation (AP-ECD) is an emerging technique for peptide analysis, suitable in principle for use with any electrospray mass spectrometer. We have recently developed a novel AP-ECD ion source utilizing nanospray for peptide ionization and photoionization of a dopant for electron generation. This source provides extensive production of c- and z-type ECD fragments from multiply-charged peptides, with detection limits in the low fmol range for samples delivered by infusion. To obtain this performance, however, manual procedures have to date been required for data collection and processing, and consequently our AP-ECD source has yet to be successfully utilized in on-line LC/MS applications. We aim to develop a fully automated AP-ECD method using our source, and then to evaluate its performance in the LC/MS analysis of protein digests.

Methods

Model proteins (e.g. BSA) will be enzymatically digested, separated by on-line LC (Ultimate LC, LC Packings), then delivered to the AP-ECD source and a Q-ToF instrument (QStar XL, AB Sciex) for mass analysis. The complete method will be automated to the greatest extent possible, to better demonstrate how it might be used in real applications. Also, as part of the performance evaluation, the data files generated by the method will be input into standard search engines (e.g. Mascot) to assess the compatibility of AP-ECD with conventional data processing tools.

Preliminary Data

To obtain high quality AP-ECD spectra of peptides with our source, it is important to eliminate background signals from nanosprayed solvent clusters/impurities, precursor "nozzle-skimmer" CID products, and photoionization by-products, since there is no precursor selection stage in-source and all the ions exiting the source contribute to the spectra generated. We have recently shown (paper submitted to JASMS) that the nanospray background continuum and CID products can be easily removed by subtracting background spectra (acquired with the photoionization lamp off) from the raw AP-ECD spectra (acquired with the lamp on). An additional subtraction step is required to remove the background of photoionization by-products, and there are a couple of ways to do this. Through these procedures, clean, low-background spectra of ECD products formed at atmospheric pressure are attainable. To date, however, the data acquisition and processing procedures required to achieve this performance have been manual, and thus impractical for on-line LC applications. Automation of the method will require a means of switching the photoionization power supply in-synch with the data acquisition method, so that ECD and background spectra can be collected alternately, continuously throughout the LC run. Implementation of this scheme is a work in progress. Once completed, the performance of the automated method will be evaluated in the LC/MS analysis of various protein digests.

Evaluation of a nanospray Atmospheric Pressure - Electron Capture Dissociation (AP-ECD) ionization source for the analysis of Post-Translational Modifications.

Davin Carter1; Jason Rogalski 1; Damon Robb2; Michael Blades2; Juergen Kast 1

1University of BC - Biomedical Research Centre, Vancouver, CANADA; 2University of British Columbia, Vancouver, BC

Novel Aspect

Hardware and method development allowing for high sensitivity ECD of post-translationally modified peptides on traditional API instruments.

Introduction

Understanding the causes and effects of many biological functions demands a mechanistic understanding of Post-Translational Modifications (PTMs) of peptides and proteins, with tandem mass spectrometry being a powerful tool in these investigations. In contrast to traditional CID, electron capture dissociation and its related technique, electron transfer dissociation, offers direct identification and localization of labile PTMs, but generally requires specialized mass spectrometers. In a previously described apparatus, photo-induced electrons were generated at atmospheric pressure to create an in-source ECD interface that could be adapted to any API mass spectrometer. A new, second generation fragmentation/ionization source allows for integration with chromatography, making it a useful addition to API mass spectrometers for tracking PTMs in proteomic investigations.

Methods

A modified AB Sciex PhotoSpray™ source was interfaced with a QStar XL™ Q-ToF so that ions from a nanospray emitter could be selectively exposed to photoelectrons from acetone to induce electron capture dissociation. With the photoionization lamp off, and no photoelectrons present, ionized peptides were admitted into the MS as in traditional nanospray - allowing conventional high sensitivity LC-MS. When the photoionization lamp was on, the resulting photoelectrons caused ECD in source and fragment ions were admitted into the mass spectrometer, allowing high sensitivity LC-AP-ECD-MS/MS. The performance of this source for the analysis of modified peptides was studied using synthetic phosphorylated, O- and N- glycoslated, sulfated and acylated peptides.

Preliminary Data

The new second generation AP-ECD source can produce nanospray sensitivity (low fmol) for peptides, and be interfaced to dissociate peptides from a chromatographic eluent stream. AP-ECD is also able to produce fragment ions with labile PTMs retained, allowing for both sensitive sequencing and localization of the PTMs, equivalent to modern ECD or ETD available on specialized instruments. The new source, however, can be incorporated in-line on any API instrument. In fact, switching to this source has enabled our Q-ToF MS to localize labile modifications on peptides at the fmol level with no need for ion trapping. For example, the O-GalNAc modification on the glycosylated peptide, HLLVSNVGGDGEEIER, is not normally directly localizable on our instrument, as CID preferentially dissociates the labile modification producing either a neutral loss of 101.5Th from the doubly charged precursor, or marker ions at 204.1Th, 186.1Th and 168.1Th. Only with the AP-ECD source are we able to directly detect and localize the modifications. A c-ion series (c4-c12) is produced in source with no evidence of cleavage of the glycosidic bond. This source has produced sensitive ECD-MS/MS spectra for singly and multiply phosphorylated peptides, in addition to peptides modified with O-linked sugars. Current studies are benchmarking the effect of dopant flows (and available photoelectrons), declustering potentials, charge state and peptide size on the c-ion yield from labile post-translationally modified peptides relative to the previous generation sources, focusing on those that are difficult to study by CID - phosphorylation, O- and N-linked sugars, sulfation and acylation.

Abstracts to CNPN ’11

Canadian National Proteomics Network, Banff

Submitted March 2011

Fusing Atmospheric Pressure ECD (AP-ECD) with nanospray to

track Post-Translational Modifications with a

"standard" mass spectrometer

Davin Carter1; Jason Rogalski1; Damon Robb2; Michael Blades2; Juergen Kast1,2

1University of BC - Biomedical Research Centre; 2University of BC - Dept. of Chemistry

Abstract

One concern of current mass spectrometry is the difficulty of analyzing labile Post- Translational Modifications (PTMs) with traditional collision induced dissociation (CID). In contrast to traditional CID, electron capture dissociation (ECD) and its related technique, electron transfer dissociation, offer direct identification and localization of labile PTMs but generally require specialized mass spectrometers. Using a modified PhotoSpray™ photoionization lamp we have recently added the capability of performing ECD to our nanospray CID Q-ToF. The modified AB Sciex PhotoSpray™ source was interfaced with a QStar XL Q-ToF so that ions from a nanospray emitter could be exposed to photoelectrons generated from acetone to induce electron capture dissociation. With the photoionization lamp off, and no photoelectrons present, ionized peptides were admitted into the MS as in traditional nanospray - allowing conventional high sensitivity LC-MS. When the photoionization lamp was on, the resulting photoelectrons caused ECD in source. C- and z-type fragment ions were admitted into the mass spectrometer, allowing high sensitivity LC-AP-ECD- MS. AP-ECD is also able to produce fragment ions with labile PTMs retained, allowing for both sequencing and localization of the PTMs, equivalent to modern ECD or ETD available on specialized instruments. The new AP-ECD source can be incorporated in-line on any atmospheric pressure ionization instrument; in fact, switching to this source has enabled our Q-ToF MS to identify labile modifications on peptides at the fmol level with no need for ion trapping.

Carter BCPN Travel Stipend Justification

(March 2011)

As a newcomer to proteomics, the CNPN conference and ETP symposium offer a fantastic opportunity to learn about the state of the art in proteomics directly from principle investigators from across Canada. I hope to make the most of the conference by attending both the hands on workshop, the structural proteomics workshop and as many sessions of the joint conference as I can. The conference will offer an in-depth introduction that will help me marry my mass spectrometry hardware design background with proteomics. I am looking forward to sharing and getting feedback on my poster "Fusing Atmospheric Pressure ECD (AP-ECD) with nanospray to track Post-Translational Modifications with a "standard" mass spectrometer." We have increased the capability of our mass spectrometer by using photoelectrons to produce ECD like spectra. I am especially interested in hearing from the proteomics community to whether they would adapt this inexpensive technology to their existing atmospheric pressure ionization spectrometers. I look forward to sharing our work, hearing what hardware development others have done and seeking input for future designs.

ASMS 2012 Abstract (submitted Feb 3, 2012)

Atmospheric Pressure Electron Capture Dissociation (AP-ECD):

Further Development and Evaluation for Localization of Labile Post-Translational Modifications on Sulfopeptides and Glycopeptides

Davin Carter 1, Jason Rogalski 1, Damon Robb 2, Michael Blades 2, Juergen Kast 1,2

1 UBC Biomedical Research Centre, 2 UBC Chemistry

Novel aspect

Localization of PTMs of sulfopeptides and glycopeptides by AP-ECD

Introduction

AP-ECD mass spectrometry is an emerging technique for analysis of labile Post-Translational Modifications. Labile PTMs are difficult to localize by traditional CID but are important for diagnosis and treatment of critical illness. In contrast to CID, ECD offers direct identification and localization of PTMs but requires specialized mass spectrometers whereas AP-ECD can be adapted to any API instrument. We have previously shown that AP-ECD is applicable at the fmol level, is effective on chromatographic time scales for mixtures and can localize modifications on phospho and glyco peptides. We have expanded our analysis to localize modifications of sulfo and additional glyco peptides and are optimizing interface energetics for our AP-ECD source.

Methods

Electrons generated from a photoionization lamp are used for Electron Capture Dissociation in a custom designed source that is interfaced with a QStar XL™. Using a Famos autosampler, samples are injected onto a lab-made C18 column and eluted using a standard peptide gradient. After the analytical column but prior to the mass spectrometer, peptides are exposed to photogenerated electrons. The electrons are produced from interactions between photons produced the photoionization lamp and a dopant, acetone. Ions are admitted into the QToF mass spectrometer where high sensitivity measurements of c and z ions take place. Simple data processing was done using Analyst.

Preliminary data

We are testing whether sulfo modifications on peptides can be localized by AP-ECD. Using caerulein, QQD(sulfo)YTGWMDF, we compared our AP-ECD results to those previously published for FT-ICR ECD. In the previous report of FT-ICR ECD analysis of Caerulein, the molecular ion and all applicable c and z ions displayed characteristic losses of 80 amu, loss of SO3. In contrast, AP-ECD spectra show that the sulfo group is retained on many c and z ions (c5-c9 & z7-z9) and on the molecular ion, allowing for localization. Another sulfo peptide was successfully analyzed and the modification localized in hirudin, DFEEIPEE(sulfo)YLQ. The sulfo modification was retained on fragment ions (c9 and c10) allowing for localization and demonstrating AP-ECD usefulness where FT-ICR ECD had failed.

We are also examining AP-ECD for glycopeptides. In previous work, we successfully used AP-ECD in the analysis of EPO (EAISPPDAA(glyco)SAAPLR) and produced near complete sequence coverage localizing the glyco modification in both c and z ions. Another glycosylated peptide, MUC5AC3 (GT(glyco)TPSPVPTTSTTSAP), was analyzed and found to retain the glycosylation on various c ion fragments but due to the structure of the peptide the modification could not be localized. A lack of basic residues and multiple prolines made ECD/ETD difficult on MUC5AC3. We will be expanding to more glycosylated peptides to demonstrate the effectiveness of AP-ECD for O-linked monosaccharides.

We aim to more fully characterize AP-ECD parameters for optimal ion transmission and to limit in-source CID of labile modifications. We have investigated the effects of varying declustering potential from 40 to 150 volts and source temperature from ambient to 150°C. Generally, larger declustering potentials improve ion transmission but cause increased CID fragmentation leading to loss of labile modifications. Similarly, increased source temperature improves ion signal but causes preferential loss of modifications.

Session: Peptides: Ion Activation/Dissociation Strategies

Abstract to BCPN conference (submitted Feb 6, 2012)

Atmospheric Pressure Electron Capture Dissociation (AP-ECD): Localization of Labile Post-Translational Modifications on Sulfopeptides

Davin Carter1; Jason Rogalski1; Damon Robb2; Michael Blades2; Juergen Kast1,2

1University of BC - Biomedical Research Centre; 2University of BC - Dept. of Chemistry

We are continuing to evaluate AP-ECD for proteomics and have recently localized sulfo modifications on peptides. AP-ECD has previously shown to be applicable at the fmol level, is effective on chromatographic time scales for mixtures and can localize modifications on phospho and glyco peptides. In contrast to traditional CID, electron capture dissociation (ECD) and its related technique, electron transfer dissociation, offer direct identification and localization of labile PTMs but generally requires specialized mass spectrometers. AP-ECD offers many of the same benefits and can be adapted to any API instrument.

Using caerulein, QQD(sulfo)YTGWMDF, we compared our AP-ECD results to those previously published for FT-ICR ECD. In the previous report of FT-ICR ECD analysis of Caerulein, the molecular ion and all applicable c and z ions displayed characteristic losses of 80 amu, loss of SO3. In contrast, AP-ECD spectra show that the sulfo group is retained on many c and z ions (c5-c9 & z7-z9) and on the molecular ion, allowing for localization. Another sulfo peptide was successfully analyzed and the modification localized in hirudin, DFEEIPEE(sulfo)YLQ. The sulfo modification was retained on fragment ions (c9 and c10) allowing for localization and demonstrating AP-ECD usefulness where FT-ICR ECD had failed.

Abstract to BCPN conference submitted Feb 6, 2012