7 research outputs found
Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-Reactive Tandem Mass Tags
Recently developed carbonyl-reactive
aminoxy tandem mass tag (aminoxyTMT)
reagents enable multiplexed characterization and quantitative comparison
of structurally complex glycans between different biological samples.
Compared to some previously reported isotopic labeling strategies
for glycans, the use of the aminoxyTMT method features a simple labeling
procedure, excellent labeling efficiency, and reduced spectral complexity
at the MS<sup>1</sup> level. Presence of the tertiary amine functionality
in the reporter region of the aminoxyTMT labels leads to increased
ionization efficiency of the labeled glycans thus improving electrospray
ionization (ESI)-mass spectrometry (MS) detection sensitivity. The
use of the labeling reagent also makes electrophoretic separation
of the labeled neutral and acidic glycans feasible. In this work,
we characterized the ESI and collision induced dissociation (CID)
behavior of the aminoxyTMT-labeled neutral and sialylated glycans.
For the high-mannose N-glycans and small sialylated oligosaccharides,
CID fragmentation of [M + Na + H]<sup>2+</sup> provides the most informative
MS<sup>2</sup> spectra for both quantitative and qualitative analysis.
For complex N-glycans, MS<sup>3</sup> of the protonated Y<sub>1(H)</sub> ion can be used for relative quantification without interference
from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS
analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides
(HMOs) and different types of N-glycans released from glycoprotein
standards were demonstrated. Improved resolution and quantification
accuracy of the labeled HMO isomers was achieved by coupling CE with
traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from
human serum protein digests were labeled with six-plex aminoxyTMT
and subjected to CE-ESI-MS/pseudo-MS<sup>3</sup> analysis, which demonstrated
the potential utility of this glycan relative quantification platform
for more complex biological samples
Targeted MultiNotch MS<sup>3</sup> Approach for Relative Quantification of N‑Glycans Using Multiplexed Carbonyl-Reactive Isobaric Tags
The recently developed
and commercially available carbonyl-reactive
tandem mass tags (aminoxyTMT) enable multiplexed quantification of
glycans through comparison of reporter ion intensities. However, challenges
still exist for collision activated dissociation (CAD) MS/MS based
quantification of aminoxyTMT due to the relatively low reporter ion
yield especially for glycans with labile structures. To circumvent
this limitation, we utilized the unique structural features of N-glycan
molecules, the common core sugar sequence (HexNAc)<sub>2</sub>(Man)<sub>3</sub>, and common <i>m</i>/<i>z</i> of Y<sub>n</sub> ions generated from different types of precursors by MS/MS
and designed a Y<sub>1</sub> ion triggered, targeted MultiNotch MS<sup>3</sup> relative quantification approach based on aminoxyTMT labeling.
This approach was implemented on a nanoHILIC-Tribrid quadrupole-ion
trap-Orbitrap platform, which enables prescreening of aminoxyTMT labeled
N-glycan precursor ions by Y<sub>1</sub> ion fragment ion mass in
a higher-energy collisional dissociation (HCD) MS/MS scan and coisolation and cofragmentation of multiple Y<sub>n</sub> fragment ions that carry the isobaric tags from the inclusion
list in the MS/MS/MS scan. Through systematical optimization and evaluation
using N-glycans released from several glycoprotein standards and human
serum proteins, we demonstrated that the Y<sub>1</sub> ion triggered,
targeted MultiNotch MS<sup>3</sup> approach offers improved accuracy,
precision, and sensitivity for relative quantification compared to
traditional data-dependent MS<sup>2</sup> and Y<sub>1</sub> ion MS<sup>3</sup> quantification methods
Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers
Quantitative
measurement of chemically cross-linked proteins is
crucial to reveal dynamic information about protein structures and
protein–protein interactions and how these are differentially
regulated during stress, aging, drug treatment, and most perturbations.
Previously, we demonstrated how quantitative in vivo cross-linking
(CL) with stable isotope labeling of amino acids in cell culture (SILAC)
enables both heritable and dynamic changes in cells to be visualized.
In this work, we demonstrate the technical feasibility of proteome-scale
quantitative in vivo CL–MS using isotope-labeled protein interaction
reporter (PIR) cross-linkers and <i>E. coli</i> as a model
system. This isotope-labeled cross-linkers approach, combined with
Real-time Analysis of Cross-linked peptide Technology (ReACT) previously
developed in our lab, enables the quantification of 941 nonredundant
cross-linked peptide pairs from a total of 1213 fully identified peptide
pairs in two biological replicate samples through comparison of MS<sup>1</sup> peak intensity of the light and heavy cross-linked peptide
pairs. For targeted relative quantification of cross-linked peptide
pairs, we further developed a PRM-based assay to accurately probe
specific site interaction changes in a complex background. The methodology
described in this work provides reliable tools for both large-scale
and targeted quantitative CL–MS that is useful for any sample
where SILAC labeling may not be practical
Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry
We recently developed
a novel amine-reactive mass-defect-based
chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative
proteomic analysis at the MS<sup>1</sup> level. In this work, we further
extend the application of the DiPyrO tag, which provides amine group
reactivity, optical detection capability, and improved electrospray
sensitivity, to quantify N-linked glycans enzymatically released from
glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ
in mass by 45.3 mDa were used to label the glycosylamine moieties
of freshly released N-glycosylamines from glycoprotein standards and
human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap
and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable
of resolving the singly or multiply charged N-glycans labeled with
mass-defect DiPyrO tags. Dynamic range of quantification, based on
MS<sup>1</sup> peak intensities, was evaluated across 2 orders of
magnitude. With optimized N-glycan release conditions, glycosylamine
labeling conditions, and MS acquisition parameters, the N-glycan profiles
and abundances in human serum proteins of cancer patients before and
after chemotherapy were compared. Moreover, this study also opens
a door for using well-developed amine-reactive tags for relative quantification
of glycans, which could be widely applied
Quantification of BSA cross-linked peptide pairs with Skyline.
<p><b>A.</b> MS2 spectrum for the cross-linked peptide pair linking residues K235-K28 (ALK<sup>235</sup>AWSVAR_DTHK<sup>28</sup>SEIAHR), obtained from a 500 ng injection of cross-linked BSA digest. <b>B.</b> Extracted ion chromatograms for the PRM transitions observed for the cross-linked peptide pair in A. <b>C.</b> Skyline generated bar plot illustrating the normalized peak areas for the cross-linked peptide pair linking K28-K235. Peak areas are shown for triplicate analyses of varying injection amounts (100, 200, 500, and 1000 ng cross-linked BSA digest). Bars are color coded to indicate the contribution of each individual transition to the total peak area and match the color scheme in panel B. </p
Cross-laboratory quantification of BSA-BDP cross-linked peptide pairs.
<p><b>A.</b> Average cross-linked peptide response curve comparing data collected in the Bruce Lab and CSHL for 30 cross-linked peptide pairs. <b>B.</b> Scatter plot illustrating the R<sup>2</sup> values for linear regression analysis of the data shown in A and B.</p
Experimental outline.
<p><b>A.</b> Biological samples are prepared for qXL-MS comparing two or more conditions. The samples are treated with chemical cross-linker either as (1) a mixed sample if SILAC labeling was used or (2) as separate samples if carrying out a label free experiment or using isotopically labeled cross-linkers. Following the cross-linking reaction proteins are extracted, enzymatically digested, and subjected to various strategies (i.e. strong cation exchange and affinity chromatography) for enrichment cross-linked peptide pairs. <b>B.</b> LC-MS analysis of samples enriched for cross-linked peptide pairs is carried out. This consists of reversed phase chromatographic separation by LC followed by analysis by MS. The mass spectrometer is operated in PRM mode where an inclusion list of <i>m/z</i> values for the precursor ions of interest is used to target specific cross-linked peptides. The PRM mass spectrometric analysis used here consists of three steps including isolation of precursor ions, fragmentation by collision with neutral gasses, and detection of mass to charge ratios of the resulting fragment ions. <b>C)</b> Resulting MS2 data are converted into transition lists and imported into Skyline for analysis.</p