skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Workflow for fast lipid tissue screening using LESA-FT-ICR-MS
Lipid screening of biological substrates is an important step during biomarker detection and identification. In this work, a fast workflow is described capable of rapid screening for lipid components from biological tissues at ambient pressure based on liquid microjunction extraction in tandem with nano-electrospray ionization (nESI) with ultra-high resolution mass spectrometry, i.e. , liquid extraction surface analysis (LESA) coupled to Fourier-transform ion cyclotron resonance (tandem) mass spectrometry (LESA-FT-ICR-MS/MS). Lipid profiles are presented for thin tissue sections of mouse brain (MB) and liver (ML) samples, analyzed in both positive and negative mode by data-dependent acquisition (DDA) tandem FT-ICR-MS/MS. Candidate assignments were based on fragmentation patterns using mostly SimLipid software and accurate mass using mostly the LipidMaps database (average sub-ppm mass error). A typical, single point surface analysis (<1 mm spatial sampling resolution) lasted less than 15 minutes and resulted in the assignment of (unique and mulitple) lipid identifications of ∼190 (MB) and ∼590 (ML) m / z values. Despite the biological complexity, this led to unique identifications of distinct lipid molecules (sub-ppm mass error) from 38 different lipid classes, corresponding to 10–30% of the lipid m / z identifications.  more » « less
Award ID(s):
1654274
PAR ID:
10128659
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Analytical Methods
Volume:
11
Issue:
18
ISSN:
1759-9660
Page Range / eLocation ID:
2385 to 2395
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract Marine dissolved organic carbon and nitrogen (DOC and DON) are major global carbon and nutrient reservoirs, and their characterization relies on extraction methods for preconcentration and salt removal. Existing methods optimize for capturing and describing DOC. Here, we report an optimized analytical strategy to recover marine DON for subsequent molecular characterization. Retention efficiencies between 5% and 95% are reported for seven solid phase extraction (SPE) sorbents, with PPL recovering 23% of marine DON compared to 95% recovered with C18. Additional comparisons of the effect of varying sample volumes and elution speed, and the resulting molecular composition of DON extracts, were investigated using C18and PPL sorbents. Sample volumes > 200 mL decreased DON retention efficiency independent of SPE sorbent, and gravity elution recovered 1.7‐ to 4.2‐fold more DON compared to vacuum elution. Characterization of extracted DON by negative‐ion electrospray ionization Fourier transform‐ion cyclotron resonance mass spectrometry (FT‐ICR MS) highlights compositional differences between DON species recovered by each method. DON isolated with optimized methods includes low molecular weight (< 600 Da) peptide‐like compounds with low O:C ratios (0.2 to 0.5) that are not detected by other SPE sorbents (e.g., PPL). The majority of additional DON isolated with this approach was undetectable by direct infusion negative mode FT‐ICR MS analysis. 
    more » « less
  2. The molecular level characterization of complex mixtures remains an analytical challenge. We have shown that the integration of complementary, high resolution, gas-phase separations allow for chemical formula level isomeric content description. In the current work, we revisited the current challenges associated with the analysis of dissolved organic matter using high resolution trapped ion mobility separation (TIMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In particular, we evaluate the separation capabilities provided by TIMS-MS compared to MS alone, the use of ICR complementary data acquisition (DAQ) systems and transient processing strategies, ICR cell geometries (e.g., Infinity cell vs harmonized cell), magnetic field strengths (7 T vs 9.4 T vs 21 T) for the case of a Harney River DOM sample. Results showed that the external high-performance DAQ enables direct representation of mass spectra in the absorption mode FT (aFT), doubling the MS resolution compared to the default magnitude mode FT (mFT). Changes between half- vs full- apodization results in greater MS signal/noise vs superior MS resolving power (RP); in the case of DOM analysis, a 45% increase in assigned formulas is observed when employing the DAQ half (Kaiser-type) apodization window and aFT when compared to the default instrument mFT. Results showed the advantages of reprocessing 2D-TIMS-FT-ICR MS data with higher RP and magnetic field chemical formulae generated list acquired (e.g., 21 T led to a 24% increase in isomers reported) or the implementation of alternative strategies. 
    more » « less
  3. In the present work, the advantages of ESI-TIMS-FT-ICR MS to address the isomeric content of dissolved organic matter are studied. While the MS spectra allowed the observation of a high number of peaks ( e.g. , PAN-L: 5004 and PAN-S: 4660), over 4× features were observed in the IMS-MS domain ( e.g. , PAN-L: 22 015 and PAN-S: 20 954). Assuming a total general formula of C x H y N 0–3 O 0–19 S 0–1 , 3066 and 2830 chemical assignments were made in a single infusion experiment for PAN-L and PAN-S, respectively. Most of the identified chemical compounds (∼80%) corresponded to highly conjugated oxygen compounds (O 1 –O 20 ). ESI-TIMS-FT-ICR MS provided a lower estimate of the number of structural and conformational isomers ( e.g. , an average of 6–10 isomers per chemical formula were observed). Moreover, ESI-q-FT-ICR MS/MS at the level of nominal mass ( i.e. , 1 Da isolation) allowed for further estimation of the number of isomers based on unique fragmentation patterns and core fragments; the later suggested that multiple structural isomers could have very closely related CCS. These studies demonstrate the need for ultrahigh resolution TIMS mobility scan functions ( e.g. , R = 200–500) in addition to tandem MS/MS isolation strategies. 
    more » « less
  4. Tandem mass spectrometry (MS/MS) is crucial for small-molecule analysis; however, traditional computational methods are limited by incomplete reference libraries and complex data processing. Machine learning (ML) is transforming small-molecule mass spectrometry in three key directions: (a) predicting MS/MS spectra and related physicochemical properties to expand reference libraries, (b) improving spectral matching through automated pattern extraction, and (c) predicting molecular structures of compounds directly from their MS/MS spectra. We review ML approaches for molecular representations [descriptors, simplified molecular-input line-entry (SMILE) strings, and graphs] and MS/MS spectra representations (using binned vectors and peak lists) along with recent advances in spectra prediction, retention time, collision cross sections, and spectral matching. Finally, we discuss ML-integrated workflows for chemical formula identification. By addressing the limitations of current methods for compound identification, these ML approaches can greatly enhance the understanding of biological processes and the development of diagnostic and therapeutic tools. 
    more » « less
  5. Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down MS. Improved sequence coverage, critical for reliable annotation of posttranslational modifications (PTMs) and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as liquid chromatography (LC)/MS of several proteins. Experiments were undertaken on multiple instruments, including Q-ToF, Orbitrap, and high-field FT-ICR across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher-energy collision dissociation (ETD-HCD) MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical vs. even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD, nor ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions. 
    more » « less