|
|
The Reid Research Group |
|
Improving the Analytical Capabilities of Biomolecular Mass
Spectrometry for Proteome and Lipidome Analysis A major goal within the emerging fields of proteomics and
lipidomics is the systematic identification, characterization and
quantitative analysis of all the biomolecules (e.g., proteins, peptides and
lipids), their co- or
post-translational modifications, and their
specific functional protein-protein or protein-lipid interactions, that are
involved in the regulation / deregulation of
normal cellular function. The outcome of
this research will enable a more complete understanding of the processes that
control cellular biochemistry, and ultimately, will enhance the
capacity of researchers in the field to identify and validate novel
biomarkers of potential therapeutic value that can subsequently be exploited
with rational drug design. To date, the application of tandem mass
spectrometry (MS/MS) based approaches to generate structural information from
the dissociation of intact peptide, protein or lipid ions formed by soft
ionization methods (ESI or MALDI), together with the development and
application of sophisticated bioinformatic tools for interrogation of the
resultant product ion spectra, have provided a powerful analytical platform
toward achieving these goals. These efforts have been underpinned by
concurrent advances in our understanding of the mechanisms and other factors
that influence the gas-phase fragmentation reactions of the various classes
of ions of interest. However, despite significant efforts over the past two
decades or more, it is fair to say that these studies are still a ‘work in
progress’, particularly with respect to understanding the influence of
process-induced or post-translational modifications on the observed
fragmentation behaviors. Thus, the current generally limited ability to (i)
accurately predict in silico the
appearance of the product ion spectra resulting from the dissociation of the
various precursor ion classes of interest, and (ii) selectively control or
direct the gas-phase fragmentation reactions of these ions toward the
formation of analytically useful products, has placed significant limitations
on the application of mass spectrometry and associated methodologies for
comprehensive proteome and lipidome analysis. Research
in the Reid laboratory at (i)
Understanding the mechanisms and other factors that influence the
gas-phase fragmentation reactions of peptide ions containing in vivo or ex
vivo modifications Determination of the
mechanisms responsible for the gas-phase fragmentation reactions of peptide
and protein ions have been the subject of significant interest over the
years. These studies have been
critical to understanding the fundamental gas-phase chemistry of these ions,
and, within the context of large scale proteomics studies, have underpinned
the development of automated MS/MS data analysis programs for sequence ion
assignment and subsequent protein identification. The mechanisms for the
formation of ‘sequence’ ions, i.e., those resulting from dissociations along
the amide backbone to yield complementary b- and y- type product ions, are
now relatively well understood. However, a common feature arising from the
low energy collision induced dissociation (CID) tandem mass spectrometry
(MS/MS) of peptide ions containing many in-vivo
or ex-vivo modifications is the
competitive formation of dominant ‘non-sequence’ ions resulting from the
loss of small molecules (e.g., RSOH, H3PO4) from the
modified amino acid side chains. Although the observation of these ‘non-sequence’
ions can be useful in providing diagnostic information regarding the presence
of the modified amino acid residue within a peptide, thereby potentially
improving the specificity of subsequent database search analysis strategies,
their formation at high abundance may ‘suppress’ the formation of desired
‘sequence’ ion information, thereby limiting the utility of ‘de-novo’
analysis strategies or current database search algorithms employed for the
identification and characterization of the modified peptide ions. Thus, it is
important to determine the conditions, such as charge state and peptide
composition (i.e., proton mobility), under which these non-sequence
fragmentation pathways are observed as dominant processes. Furthermore, given
that MS3 dissociation of abundant non-sequence product ions is
often employed to obtain additional sequence information to facilitate
further structural characterization, it is also important to determine the
mechanisms responsible for their formation, and the structures of the
resultant product ions. Beginning
with independent research carried out from 2002-2004 at the Ludwig Institute
for Cancer Research, and continuing at Michigan State University since August
2004, we have been working toward developing an improved understanding of the
mechanisms and other factors (e.g., peptide ion charge state, amino acid
composition, site specific amino acid location, or peptide conformation) that
influence the formation and relative abundance of ‘sequence’ versus ‘non-sequence’
ions from the fragmentation of peptide ions containing a range of in-vivo (phosphorylation) and ex-vivo (oxidation, alkylation,
chemically cross-linked) modifications. Kapp, E.A., Schütz, F., Reid, G.E., Eddes, J.S., Moritz, R.L., O’Hair,
R.A.J., Speed, T.P. and Simpson, R.J. (2003) Mining a
tandem mass spectrometry database to determine the trends and global factors
influencing peptide fragmentation. Anal.
Chem. 75: 6251-6264. Hohmann, L.J., Eng, J.K., Gemmill,
A., Klimek, J., Vitek, O., Reid, G.E. and Martin D. B. (2008) Quantification of the Compositional Information
Provided by Immonium Ions on a Quadrupole-TOF Mass Spectrometer. Anal. Chem.
80: 5596-5606. Lioe, H., O’Hair, R.A.J. and Reid, G.E. (2004) A Mass
Spectrometric and Molecular Orbital Study of H2O Loss from
Tryptophan and Oxidized Tryptophan Derivatives. Rapid Commun. Mass Spectrom.
18: 978-988. Lioe, H., O’Hair, R.A.J. and Reid, G.E. (2004) Gas-Phase
Reactions of Protonated Tryptophan. J.
Am. Soc. Mass Spectrom. 15: 65-76. Oxidation Oxidation is one of the more common ex-vivo (i.e, process-induced)
modifications encountered during the sequence analysis of proteins by tandem
mass spectrometry. We have shown, by statistical analysis of a large database
of methionine sulfoxide containing peptide product ion spectra indicated that
the structurally diagnostic ‘non-sequence ion’ neutral loss of methane
sulfenic acid (CH3SOH, 64Da) from the side chain of methionine
sulfoxide residues is the dominant fragmentation process ions under
conditions of low proton mobility, i.e., when ionizing proton(s) are
sequestered at strongly basic amino acids such as arginine, lysine or
histidine. Multistage MS/MS of several methionine sulfoxide containing model
‘tryptic’ peptides, combined with regioselective structural and isotopic
labeling, independent solution phase chemical synthesis of proposed gas-phase
product ions and ab initio
molecular orbital calculations, indicated that the pathway for this loss
proceeded via a ‘charge remote’ cis-elimination mechanism process. Reid, G.E., Roberts, K.D., Kapp, E.A. and Simpson, R.J.
(2004) Statistical and Mechanistic Approaches to Understanding the Gas-phase
Fragmentation Behaviour of Methionine Sulfoxide Containing Peptides. J. Prot.
Res. 3: 751-759. Further insights into the factors controlling the
fragmentation behavior of methionine sulfoxide containing peptides have been
obtained from the results of a theoretical and experimental study (in
collaboration with Professors Scott Gronert ( Lioe, H., Laskin, J., Reid, G.E. and O’Hair, R.A.J. (2007)
Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides
containing Methionine Sulfoxide or Aspartic Acid via Energy- and
Time-Resolved Surface Induced Dissociation. J. Phys. Chem. A. 111: 10580-10588. Lioe, H., O'Hair, R.A.J. Gronert, S., Austin, A. and Reid,
G.E. (2007) Experimental and Theoretical proton affinities of Methionine,
Methionine sulfoxide and their N- and C-terminal derivatives. Int. J. Mass
Spectrom. 267: 220-232. Other
studies have also been carried out to examine the mechanisms responsible for
the ‘non-sequence’ fragmentation reactions of S-alkyl cysteine sulfoxide
containing peptides prepared by reaction with the common alkylating reagents
iodomethane, iodoacetamide, iodoacetic acid, acrylamide or 4-vinylpyridine,
then followed by oxidation with hydrogen peroxide, using multistage tandem
mass spectrometry, hydrogen/deuterium exchange and molecular orbital
calculations (at the B3LYP/6-31+G(d,p) level of theory). Consistent with
earlier reports in the literature, CID-MS/MS of the S-alkyl cysteine
sulfoxide-containing peptide ions results in the dominant ‘non-sequence’
neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side
chains under conditions of low proton mobility, irrespective of the
alkylating reagent employed. However, the
mechanisms responsible for this loss had not previously been determined.
Dissociation of uniformly deuterated peptide precursor ions was employed to
determine that the mechanism for loss of alkyl sulfenic acid in each case
occurred via a ‘charge-remote’ five-centered cis-1,2 elimination reaction to yield a dehydroalanine containing
product ion. Similarly, the charge
state dependence to the mechanisms and product ion structures for the losses
of CO2, CO2+H2O and CO2+CH2O
from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the
losses of CH2CHCONH2 and CH2CHC5H4N,
respectively from S-amidoethyl and S-pyridylethyl cysteine
sulfoxide-containing peptide ions have also been determined. The results from these studies
indicate that both the proton mobility of the peptide precursor ion and the
nature of the S-alkyl substituent have a significant influence on the
abundances and charge states of the product ions resulting from the various
competing fragmentation pathways. Froelich J.M. and Reid, G.E. (2007) Mechanisms for the Proton Mobility
Dependant Gas-Phase Fragmentation Reactions of S-alkyl Cysteine
Sulfoxide-containing Peptide Ions. J. Am. Soc. Mass Spectrom. 18: 1690-1705. We have also directed efforts toward determining the
conditions for selectively controlling oxidative peptide modifications prior
to their analysis by tandem mass spectrometry, and to evaluate the effect of
‘non-sequence’ fragmentations resulting from oxidative modifications on the
efficacy of database search algorithms for automated peptide identification
and characterization. The origin of increased oxidation levels were found to
be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis
and/or in-gel proteolytic digestion of proteins prior to analysis by mass
spectrometry. Subsequently, conditions for deliberately controlling the
oxidation state (either oxidation or reduction) of these peptides have been
determined. Quantitative analysis of the product ion abundances within the
spectra obtained from the fragmentation of a series of methionine sulfoxide
or S-alkyl cysteine sulfoxide containing peptides clearly indicates that an
increase in the abundance of the ‘non-sequence’ product ions corresponding to
the characteristic ‘non-sequence’ side chain neutral loss of RSOH from these
peptides results in a corresponding decrease in the magnitude of the database
search scores, and in some cases, results in an inability to identify the
peptide by database search methods. Froelich, J.M. and Reid, G.E. (2008). The Origin and
Control of Ex Vivo Oxidative Peptide Modifications Prior to Mass Spectrometry
Analysis. Proteomics. 8: 1334-1345. Phosphorylation The development of strategies directed toward comprehensive
analysis of the phosphoproteome have undoubtedly been facilitated by recent
advances in the application of ion trap tandem mass spectrometry-based
techniques for routine phosphopeptide identification. However, when multiple
potential sites of phosphorylation exist within a phosphorylated peptide
sequence, unambiguous characterization of the site of phosphorylation remains
a significant challenge. Recently, we
have carried out a series of
fundamental studies to systematically determine the mechanisms and
other factors that influence the multistage gas-phase fragmentation reactions
of phosphoserine and phosphothreonine containing peptides. From this study,
it was found that the magnitude of
product ions formed via the neutral loss of 98 Da (typically assigned
as phosphoric acid (H3PO4)) were highly dependent on the proton mobility of the precursor ion. A mechanism for this loss,
involving a ‘charge remote’ pathway had previously been accepted in the literature for a period of over 10
years. However, using regioselective and
uniformly deuterium labeled peptides, as well as MS3 of the initial [M+nH-98]n+
product ions, our data clearly indicated that ‘charge-directed’
mechanisms were responsible for the observed fragmentation behavior. Furthermore, the observation of product ions corresponding
to the loss of formaldehyde (CH2O, 30 Da or CD2O, 32
Da) or acetaldehyde (CH3CHO, 44 Da) upon MS3
dissociation of the [M+nH-98]n+ product ions from phosphoserine-
and phosphothreonine-containing peptide ions, respectively, provided direct
experimental evidence for an SN2 neighboring group participation
reaction, resulting in the formation of a cyclic product ion. It was initially hoped that these
‘diagnostic’ MS3 product ions could be employed to provide
additional information to facilitate the characterization of phosphopeptides
containing multiple potential phosphorylation sites. However, upon examining
the collision CID-MS/MS and -MS3 fragmentation reactions of a
series of independently synthesized phospho-serine, -threonine and -tyrosine
peptides containing multiple potential phosphorylation sites in a linear
quadrupole ion trap, it was unexpectedly found that 45% of the peptides gave
rise to MS/MS and/or MS3 product ions that indicated the site of
phosphorylation was located at the ‘incorrect’ position. The origin of the
‘erroneous’ MS/MS product ions were found to arise from an initial gas-phase
transfer of a phosphate group from the phosphorylated residue to an
unmodified hydroxyl-containing amino acid residue upon CID-MS/MS, but prior
to peptide dissociation. The propensity for this rearrangement was found to
be highly dependant on the precursor ion charge state and amino acid
composition (i.e, proton mobility) of the peptide, and was observed
predominantly for peptides under ‘non-mobile’ or ‘partially-mobile’
protonation conditions. ‘Erroneous’ MS3 product ions could be
formed due to competing fragmentation reactions for the neutral loss of 98 Da
from these precursor ions (i.e., the loss of H3PO4 versus
the combined losses of HPO3 and H2O), indicating that
CID-MS3 of [M+nH-98]n+ ions
may not be used for unambiguous phosphorylation site localization. Importantly, the observation of these rearrangement
reactions, and/or the lack of product ions that provide definitive evidence
for the correct site of phosporylation, was found to limit the ability to unambiguously assign the
correct site of phosphorylation to only 36% of the peptides. Palumbo, A.M., Tepe, J.J. and
Reid, G.E. (2008) Mechanistic Insights into the Multistage Gas-Phase
Fragmentation Behavior of Phosphoserine- and Phosphothreonine-containing
Peptides. J. Proteome Res. 7:
771-779. Palumbo, A.M and Reid, G.E. (2008)
Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions
on Protein Phosphorylation Site Assignment using CID-MS/MS and MS3.
Anal. Chem. In Press. Dunn, J.D., Igrisan, E.A., Palumbo, A.M., Reid, G.E. and
Bruening, M.L. (2008) Phosphopeptide Enrichment Using MALDI Plates Modified
with High-capacity Polymer Brushes. Anal. Chem. 80: 5727-5735. Froelich, J.M. and Reid, G.E. (2008) The Effect of
Post-translational and Process-induced Modifications on the Multistage
Gas-Phase Fragmentation Reactions of Protonated Peptide Ions. Combinatorial
Chemistry and High Throughput Screening. In Press. Taken together, the above studies clearly
demonstrate the role of peptide modifications on fragmentation behavior, and
highlights the need to develop improved database search methods for peptide
identification and characterization that incorporate the fragmentation
‘rules’ arising from this work. (ii)
Chemical
Methods and Tandem Mass Spectrometry Strategies for ‘Targeted’ Proteome
Analysis As a direct result of the
improved mechanistic understanding of ‘non-sequence’ ion peptide
fragmentation reactions obtained above, we have developed a novel strategy
for selective protein identification and differential quantitative analysis,
termed 'Selected Extraction of Labelled
Entities by Charge derivatization and Tandem mass spectrometry' (SELECT).
This strategy involves the introduction of a 'fixed-charge' sulfonium ion to
peptides or proteins containing certain structural features (e.g., the side
chains of selected amino acids). MS/MS of these peptide ions results in
exclusive loss of the derivatized side chain and the formation of a single
characteristic product ion, independently of the peptide ion charge state or
amino acid composition. Thus, fixed
charge containing peptide ions may be selectively identified from complex
mixtures by, for example, automated selective neutral loss or precursor ion
scan mode MS/MS methods, without requirement for purification or otherwise
enrichment prior to analysis, and with an increase in selectively and
sensitivity of several orders of magnitude over existing MS based approaches.
Further structural interrogation of identified peptide ions is readily
achieved by subjecting the characteristic MS/MS product ion to multistage
MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by
energy resolved 'pseudo' MS3 in a triple quadrupole mass
spectrometer The general principles underlying this fixed charge
derivatization approach have been demonstrated by MS/MS, MS3 and
'pseudo' MS3 analysis of side chain fixed-charge sulfonium ion
derivatives of peptides containing methionine and cysteine. Furthermore, the
incorporation of 'light' and ‘heavy’ isotopically encoded labels into the
fixed-charge derivatives has also enabled the application of this approach to
the quantitative analysis of differential protein expression, via measurement
of the relative abundances of the neutral loss product ions generated by
dissociation of the light and heavy labelled peptide ions. Reid, G.E.,
Roberts, K.D., Simpson, R.J., O'Hair, R.A.J. (2005) Selective Identification
and Quantitative Analysis of Methionine Containing Peptides by Charge
derivatization and Tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 16:
1131-1150. Froelich, J.M., Kaplinghat, S. and Reid, G.E. (2008)
Automated Neutral Loss and Data Dependent Energy Resolved “Pseudo MS3”
for the Targeted Identification, Characterization and Quantitative Analysis
of Methionine-Containing Peptides. Eur. J. Mass Spectrom. 14: 219-229. Roberts, K.D. and Reid,
G.E. (2007) Leaving Group Effects on the Selectivity of
the Gas-Phase Fragmentation Reactions of Side Chain Fixed Charge Containing
Peptide Ions. J. Mass Spectrom. 42: 187-198. Froelich, J.M., Lu, Y. and Reid, G.E. (2008) Chemical
Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural
Characterization. In: Practical Aspects of Trapped Ion Mass Spectrometry.
Vol. 5: Applications. (R.E. March and J.F.J. Todd. Ed), CRC Press. In Press. The
mechanisms responsible for the fragmentation of 'fixed-charge'
phenacylsulfonium ion derivatized methionine containing peptide ions have
been determined, by experimental evidence obtained from multistage
dissociation of a regioselectively deuterated methionine derivatized
sulfonium ion containing tryptic peptide, as well as by molecular orbital
calculations performed on a simple peptide model, to occur via SN2
reactions involving the N-and C-terminal amide bonds adjacent to the
methionine side chain, resulting in the formation of stable cyclic five- and
six-membered iminohydrofuran and oxazine product ions, respectively. These
studies further indicated that the rings formed via these neighboring group
reactions are stable to further dissociation by MS3. The effect of
various para-substituents on the multistage (MS/MS and MS3)
fragmentation reactions of the methionine side chain fixed charge
phenacylsulfonium ion containing peptides have also been examined. Loss of
the methylphenacylsulfide side chain fragment as neutral versus protonated
species were observed to be dependent on the proton mobility of the precursor
ion and the identity of the para-substituent. The log of the ratio of neutral
versus charged losses of the derivatized side chain were found to exhibit a
linear dependence on the proton affinity of the side chain fragmentation
product, as well as the proton affinities of the peptide product ions. Amunugama, M., Roberts, K.D. and Reid,
G.E. (2006) Mechanisms for the Selective Gas-phase Fragmentation
Reactions of Methionine Side Chain Fixed Charge Sulfonium Ion Containing
Peptides. J. Am. Soc. Mass Spectrom. 17: 1631-1642. Sierakowski, J., Amunugama, M., Roberts, K.D and Reid, G.E. (2007) Substituent
Effects on the Gas-Phase Fragmentation Reactions of Sulfonium Ion Containing
Peptides. Rapid Commun.
Mass Spectrom. 21: 1230-1238. More recently, this
sulfonium ion derivatization approach has also been extended toward the
development of an improved MS/MS based analysis strategy for the
characterization of protein structure, protein folding and protein-protein
interactions. Chemical cross-linking combined with proteolytic digestion and
mass spectrometry (MS) is a promising approach to provide inter- and
intramolecular distance constraints for the structural characterization of
protein topologies and functional multi-protein complexes.
Despite the
relative straightforwardness of these methodologies, the identification and
characterization of cross-linked proteins presents a significant analytical
challenge, due to the complexity of the resultant peptide mixtures, as well as
the array of
inter-, intra- or “dead-end”- cross-linked peptides that may be generated
from a single cross-linking experiment. To address these issues,
we recently described the synthesis, characterization and initial evaluation
of a novel “fixed
charge” sulfonium ion containing cross-linking reagent, S-methyl
5,5’-thiodipentanoylhydroxysuccinimide. The peptide products obtained by reaction with this
reagent are all shown to fragment exclusively via facile cleavage of the C-S
bond directly adjacent to the fixed charge during CID-MS/MS, resulting in the
formation of characteristic products ions that enable the presence and type
(i.e., inter-, intra- or dead-end) of the cross-linked products to be readily
determined, independently of the “proton mobility” of the
precursor ion.
Subsequent isolation and dissociation of these products by MS3
provides additional structural information required for identification of the
peptide sequences involved in the cross-linking reactions, as well as for
characterization of the specific site(s) at which cross-linking has occurred.
The specificity of these gas-phase fragmentation reactions, as well as the
solubility and stability of the cross-linking reagent under aqueous
conditions, suggest that this strategy holds great promise for use in future
studies aimed at the structural analysis of large proteins or multi-protein
assemblies. Lu, Y., Tanasova, M., Borhan, B. and Reid, G.E. (2008) An Ionic Reagent for Controlling the
Gas-Phase Fragmentation Reactions of Cross-Linked Peptides. Anal. Chem.
In Press. (iii) Top Down Protein Characterization The key advantage of ‘top-down’ approaches to
protein identification and characterization, involving the dissociation of intact
protein ions rather than their 'bottom-up' proteolytically derived peptides,
is that the entire sequence of the protein is available for interrogation,
thereby enabling protein identification to be potentially be achieved in a
single step, including the characterization of any co- or post-translational
modifications. Reid, G.E. Characterization of
Proteins by Mass Spectrometry. (2003) In: 'Purifiying Proteins: A Laboratory Manual' (Simpson, R.J. Ed.)
We have carried out a study to determine the role
of the active site residues within the Staphylococcus
aureus Dihydroneopterin aldolase (SaDHNA) enzyme by ‘top-down’ tandem
mass spectrometry methods. In this study, the gas-phase fragmentation
reactions of a series of active-site directed mutagenesis products of SaDHNA
have been examined in order to (i) evaluate the utility of a linear
quadrupole ion trap mass spectrometer for routine 'top-down' recombinant
protein characterization, (ii) examine the precursor ion charge state
dependence on the gas-phase fragmentation behavior of these proteins, and
(iii) confirm the mutation sites. Although proving to be successful for the
majority of the protein mutants examined, this work highlighted the need for
further studies to characterize the charge state, sequence and structural
dependence to the fragmentation behavior of multiply protonated intact
protein ions, and will form the basis for further efforts in our laboratory
in the future. Scherperel, G., Yan, H., Wang, Y.
and Reid, G.E. (2006) Characterization of dihydroneopterin aldolase site
directed mutagenesis products from Staphylococcus Aureus by 'top down'
multistage tandem mass spectrometry in a linear quadrupole ion trap. The
Analyst. 131: 291-302. This work has been extended toward the analysis of
the structure-function role that a conserved active-site Y61 residue plays
within the DHNA sequence, by the identification of a novel product arising
from the reaction of the Y61F mutant protein with the enzyme substrate
7,8-dihydroneopterin (DHNP). This result led to the proposal that the active
site Y61F mutant acts as an oxygenase rather than an aldolase. Wang, Y., Scherperel, G., Roberts,
K.D., Jones, A.D., Reid, G.E. and Yan, H. (2006) A Point Mutation Converts Dihydroneopterin Aldolase to a
Cofactor-Independent Oxygenase. J. Am. Chem. Soc. 128: 13216-13223. A short ‘educational’ overview of the current strategies employed for
top-down protein characterization, and the key technical challenges and
solutions associated with their implementation on a range of mass
spectrometry instrument platforms, was published as an invited review article
to the Education i-section of The
Analyst. Scherperel, G. and Reid, G.E. (2007) Emerging methods in
Proteomics: Multistage Tandem Mass Spectrometry for Top-down Protein
Characterization. The Analyst. 132: 500 –
506. (iv) Identification and Characterization of
Lipids by Multistage Tandem Mass Spectrometry. The identification of biomarkers that enable the early
detection and prognosis of disease, or that facilitate measurement of the
efficacy of response to a specific therapeutic intervention, holds great
promise in advancing the capabilities of individualized medicine. Recent technological advances
in mass spectrometry have enabled large scale biomarker discovery efforts to
be initiated, including in the field of lipidomics, without prior requirement
for detailed insights into the mechanisms responsible for the disease. Lipids are a diverse group of compounds,
including fatty acyls, sterols,
glycerolipids, glycerophospholipids and sphingolipids,
that play key biological roles as the main structural component of
cell membranes, in energy storage and metabolism,
and in cell signaling. A large number
of studies have demonstrated that the disruption of lipid metabolism or
signaling pathways can play a key role in the onset and progression of human
disease, including diabetes, diabetic complications and cancer. In
some instances, monitoring changes in the abundance of particular lipid
species between diseased and normal tissue has been shown to provide a
greater ability to detect the disease at an earlier stage of progression
compared to conventional protein biomarkers. A
necessary prerequisite to the quantitative mass spectrometry based
characterization of changes in lipid profiles that occur as a function of the
onset and progression of disease in a particular cell, tissue or organ, is to
first develop effective strategies for identification of the individual lipid
components that may be present, without need for extensive sample handling or
fractionation. To address this
requirement, we have initiated a series of studies to systematically examine
the gas-phase fragmentation reactions of various lipid classes that may be
observed in the mass spectrometer in various ionic forms, from within a
complex crude lipid extracts Zhang, X., and Reid, G.E. (2006)
Multistage Tandem Mass Spectrometry of Anionic Phosphatidylcholine Lipid
Adducts Reveals Novel Dissociation Pathways. Int. J. Mass Spectrom. 252:
242-255. Zhang, X., Ferguson-Miller, S.M. and Reid, G.E. (2009) Characterization of Ornithine and
Glutamine Lipids Extracted from Cell Membranes of Rhodobacter sphaeroides. J.
Am. Soc. Mass Spectrom. In Press. doi:10.1016/j.jasms.2008.08.017 The results from these
and related fundamental gas-phase lipid ion chemistry studies are now being
applied toward the development of comprehensive ‘shotgun’ MS/MS based
approaches using multiple lipid-class specific precursor
ion and neutral loss scan mode experiments in a triple quadrupole mass
spectrometer, or multistage (MSn)
tandem mass spectrometry in a quadrupole ion trap mass spectrometer, to (i) identify and quantify the role of integral lipids
in the structure and function of the membrane protein complex, cytochrome c
oxidase (in collaboration with Prof. Shelagh Ferguson-Miller, Dept. Biochem.
and Mol. Biol. MSU), and (ii) to identify and quantify the temporal changes in lipid profiles associated with the onset of diabetic
retinopathy in rat models of type 1 diabetes
(in collaboration with Prof. Julia Busik, Dept.
Physiol. MSU) and the development of hepatocellular
carcinoma in a transgenic mouse model (in
collaboration with Prof. Rheal Towner, Oklahoma Medical Research
Foundation, Oklahoma). Griffitts, J., Tesiram, Y., Reid, G.E., Saunders, D., Floyd, R. and
Towner, R. (2009) In Vivo magnetic
resonance spectroscopy (MRS) assessment of altered fatty acyl unsaturation in
liver tumor formation of a TGFα/c-myc transgenic mouse model. J. Lipid.
Res. In Press. |
|
|
|
|
|
This page maintained by Gavin
Reid. Last Updated: October 18th, 2008 |