9 research outputs found
Recommendations for reporting ion mobility mass spectrometry measurements
Ā© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. Ā© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc
Ion MobilityāMass Spectrometry of Complex Carbohydrates: Collision Cross Sections of Sodiated Nālinked Glycans
Currently, the vast majority of complex
carbohydrates are characterized
using mass spectrometry (MS)-based techniques. Measuring the molecular
mass of a sugar, however, immediately poses a fundamental problem:
entire classes of the constituting monosaccharide building blocks
exhibit an identical atomic composition and, consequently, also an
identical mass. Therefore, carbohydrate MS data can be highly ambiguous
and often it is simply not possible to clearly assign a particular
molecular structure. A promising approach to overcome the above-mentioned
limitation is to implement an additional gas-phase separation dimension
using ion mobility spectrometry (IMS), which is a method in which
molecules of identical mass and structure but different structure
can be separated according to their shape and collision cross section
(CCS). With the emergence of commercially available hybrid ion mobilityāmass
spectrometry (IM-MS) instruments in 2006, IMS technology became readily
available. Because of the nonhomogeneous, traveling wave (TW) field
utilized in these instruments, however, CCS values currently cannot
be determined directly from the drift times measured. Instead, an
external calibration using compounds of known CCS and similar molecular
identity is required. Here, we report a calibration protocol for TW
IMS instruments using a series of sodiated <i>N</i>-glycans
that were released from commercially available glycoproteins using
an easy-to-follow protocol. The underlying CCS values were determined
using a modified Synapt HDMS instrument with a linear drift tube,
which was described in detail previously. Our data indicate that,
under in-source fragmentation conditions, only a few glycans are required
to obtain a TW IMS calibration of sufficient quality. In this context,
however, the type of glycan was shown to be of tremendous importance.
Furthermore, our data clearly demonstrate that carbohydrate isomers
with identical mass but different conformation can be distinguished
based on their CCS when all the associated errors are taken into account
Labeling of Mucin-Type <i>O</i>āGlycans for Quantification Using Liquid Chromatography and Fluorescence Detection
O-glycosylation is a common post-translational
modification that is essential for the defensive properties of mucus
barriers. Incomplete and altered O-glycosylation
is often linked to severe diseases, such as cancer, cystic fibrosis,
and chronic obstructive pulmonary disease. Originating from a nontemplate-driven
biosynthesis, mucin-type O-glycan structures are
very complex. They are often present as heterogeneous mixtures containing
multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques
such as liquid chromatography (LC), ion mobility spectrometry, and
mass spectrometry (MS). However, MS-based techniques are mainly qualitative.
Moreover, LC separation of O-glycans often lacks
reproducibility and requires sophisticated data treatment and analysis.
Here we present a mucin-type O-glycomics analysis
workflow that utilizes hydrophilic interaction liquid chromatography
for separation and fluorescence labeling for detection and quantification.
In combination with mass spectrometry, a detailed analysis on the
relative abundance of specific mucin-type O-glycan
compositions and features, such as fucose, sialic acids, and sulfates,
is performed. Furthermore, the average number of monosaccharide units
of O-glycans in different samples was determined.
To demonstrate universal applicability, the method was tested on mucins
from different tissue types and mammals, such as bovine submaxillary
mucins, porcine gastric mucins, and human milk mucins. To account
for day-to-day retention time shifts in O-glycan
separations and increase the comparability between different instruments
and laboratories, we included fluorescently labeled dextran ladders
in our workflow. In addition, we set up a library of glucose unit
values for all identified O-glycans, which can be
used to simplify the identification process of glycans in future analyses
Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific <i>Cis</i>/<i>Trans</i> Isomerization of Proline Peptide Bonds
Ultraviolet
photodissociation (UVPD) of gas-phase proteins has
attracted increased attention in recent years. This growing interest
is largely based on the fact that, in contrast to slow heating techniques
such as collision induced dissociation (CID), the cleavage propensity
after absorption of UV light is distributed over the entire protein
sequence, which can lead to a very high sequence coverage as required
in typical top-down proteomics applications. However, in the gas phase,
proteins can adopt a multitude of distinct and sometimes coexisting
conformations, and it is not clear how this three-dimensional structure
affects the UVPD fragmentation behavior. Using ion mobilityāUVPDāmass
spectrometry in conjunction with molecular dynamics simulations, we
provide the first experimental evidence that UVPD is sensitive to
the higher order structure of gas-phase proteins. Distinct UVPD spectra
were obtained for different extended conformations of 11<sup>+</sup> ubiquitin ions. Assignment of the fragments showed that the majority
of differences arise from <i>cis/trans</i> isomerization
of one particular proline peptide bond. Seen from a broader perspective,
these data highlight the potential of UVPD to be used for the structural
analysis of proteins in the gas phase
Amide-I and -II Vibrations of the Cyclic Ī²-Sheet Model Peptide Gramicidin S in the Gas Phase
In the condensed phase, the peptide gramicidin S is often considered
as a model system for a Ī²-sheet structure. Here, we investigate
gramicidin S free of any influences of the environment by measuring
the mid-IR spectra of doubly protonated (deuterated) gramicidin S
in the gas phase. In the amide I (i.e., Cī»O stretch) region,
the spectra show a broad split peak between 1580 and 1720 cm<sup>ā1</sup>. To deduce structural information, the conformational space has
been searched using molecular dynamics methods and several structural
candidates have been further investigated at the density functional
level. The calculations show the importance of the interactions of
the charged side-chains with the backbone, which is responsible for
the lower frequency part of the amide I peak. When this interaction
is inhibited via complexation with two 18-crown-6 molecules, the amide
I peak narrows and shows two maxima at 1653 and 1680 cm<sup>ā1</sup>. A comparison to calculations shows that for this complexed ion,
four Cī»O groups are in an antiparallel Ī²-sheet arrangement.
Surprisingly, an analysis of the calculated spectra shows that these
Ī²-sheet Cī»O groups give rise to the vibrations near 1680
cm<sup>ā1</sup>. This is in sharp contrast to expectations
based on values for the condensed phase, where resonances of Ī²-sheet
sections are thought to occur near 1630 cm<sup>ā1</sup>. The
difference between those values might be caused by interactions with
the environment, as the condensed phase value is mostly deduced for
Ī²-sheet sections that are embedded in larger proteins, that
interact strongly with solvent or that are part of partially aggregated
species
Estimating Collision Cross Sections of Negatively Charged <i>N-</i>Glycans using Traveling Wave Ion Mobility-Mass Spectrometry
Glycosylation is one of the most
common post-translational modifications
occurring in proteins. A detailed structural characterization of the
involved carbohydrates, however, is still one of the greatest challenges
in modern glycoproteomics, since multiple regio- and stereoisomers
with an identical monosaccharide composition may exist. Recently,
ion mobility-mass spectrometry (IM-MS), a technique in which ions
are separated according to their mass, charge, and shape, has evolved
as a promising technique for the separation and structural analysis
of complex carbohydrates. This growing interest is based on the fact
that the measured drift times can be converted into collision cross
sections (CCSs), which can be compared, implemented into databases,
and used as additional search criteria for structural identification.
However, most of the currently used commercial IM-MS instruments utilize
a nonuniform traveling wave field to propel the ions through the IM
cell. As a result, CCS measurements cannot be performed directly and
require calibration. Here, we present a calibration data set consisting
of over 500 reference CCSs for negatively charged <i>N</i>-glycans and their fragments. Moreover, we show that dextran, already
widely used as a calibrant in high performance liquid chromatography,
is also a suitable calibrant for CCS estimations. Our data also indicate
that a considerably increased error has to be taken into account when
reference CCSs acquired in a different drift gas are used for calibration
Secondary Structure of Ac-Ala<sub><i>n</i></sub>-LysH<sup>+</sup> Polyalanine Peptides (<i>n</i> = 5,10,15) in Vacuo: Helical or Not?
The polyalanine-based peptide series Ac-Ala<sub><i>n</i></sub>-LysH<sup>+</sup> (<i>n</i> = 5ā20) is a prime example that a secondary structure motif that is well-known from the solution phase (here: helices) can be formed in vacuo. Here we revisit the series members <i>n</i> = 5,10,15, using density functional theory (van der Waals corrected generalized gradient approximation) for structure predictions, which are then corroborated by room temperature gas-phase infrared vibrational spectroscopy. We employ a <i>quantitative</i> comparison based on Pendryās reliability factor (popular in surface crystallography). In particular, including <i>anharmonic</i> effects into calculated spectra by way of ab initio molecular dynamics produces remarkably good experimentātheory agreement. We find the longer molecules (<i>n</i> = 10,15) to be firmly Ī±-helical in character. For <i>n</i> = 5, calculated free-energy differences show different H-bond networks to still compete closely. Vibrational spectroscopy indicates a predominance of Ī±-helical motifs at 300 K, but the lowest-energy conformer is not a simple helix
Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest
In recent years, glycosaminoglycans (GAGs) have emerged
into the
focus of biochemical and biomedical research due to their importance
in a variety of physiological processes. These molecules show great
diversity, which makes their analysis highly challenging. A promising
tool for identifying the structural motifs and conformation of shorter
GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this
work, the cryogenic gas-phase IR spectra of mass-selected heparan
sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to
extract vibrational features that are characteristic to structural
motifs. The data were augmented with chondroitin sulfate (CS) disaccharide
spectra to assemble a training library for random forest (RF) classifiers.
These were used to discriminate between GAG classes (CS or HS) and
different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With
optimized data preprocessing and RF modeling, a prediction accuracy
of >97% was achieved for HS tetra- and hexasaccharides based on
a
training set of only 21 spectra. These results exemplify the importance
of combining gas-phase cryogenic IR ion spectroscopy with machine
learning to improve the future analytical workflow for GAG sequencing
and that of other biomolecules, such as metabolites
Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels
Solubilized, gel-forming
decellularized extracellular matrix (dECM)
is used in a wide range of basic and translational research and due
to its inherent bioactivity can promote structural and functional
tissue remodeling. The animal-derived protease pepsin has become the
standard proteolytic enzyme for the solubilization of almost all types
of collagen-based dECM. In this study, pepsin was compared with papain,
Ī±-amylase, and collagenase for their potential to solubilize
porcine liver dECM. Maximum preservation of bioactive components and
native dECM properties was used as a decisive criterion for further
application of the enzymes, with emphasis on minimal destruction of
the protein structure and maintained capacity for physical thermogelation
at neutral pH. The solubilized dECM digests, and/or their physically
gelled hydrogels were characterized for their rheological properties,
gelation kinetics, GAG content, proteomic composition, and growth
factor profile. This study highlights papain as a plant-derived enzyme
that can serve as a cost-effective alternative to animal-derived pepsin
for the efficient solubilization of dECM. The resulting homogeneous
papain-digested dECM preserved its thermally triggered gelation properties
similar to pepsin digests, and the corresponding dECM hydrogels demonstrated
their enhanced bioadhesiveness in single-cell force spectroscopy experiments
with fibroblasts. The viability and proliferation of human HepaRG
cells on dECM gels were similar to those on pure rat tail collagen
type I gels. Papain is not only highly effective and economically
attractive for dECM solubilization but also particularly interesting
when digesting human-tissue-derived dECM for regenerative applications,
where animal-derived materials are to be avoided