Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8-355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples.

This chapter demonstrates how key mass spectrometry technologies can coalesce into straightforward, accurate, extremely sensitive, and, most importantly, high-throughput quantitative solutions. It presents an extensive resource composed of peptide quantitation studies performed on mass spectrometry instruments. Also, the chapter illustrates how sensitive and selective detection can be achieved even in the presence of high background noise. It then details advances in high-resolution mass spectrometry, which highlights targeted workflows on the high-resolution, accurate-mass spectrometry (HRMS) system that extend the sensitivity and selectivity of quantitative assays due to the narrow extraction widths and high-resolution time-of-flight (TOF) data. The chapter further investigates the software tools available for robust peptide quantitation workflows that give researchers intuitive tools to automate the complex, multistep calculations for peak-area quantitation. Finally, it focuses on experiments conducted on two types of hybrid triple quadrupole instruments: high-resolution, accurate-mass spectrometry quadrupole TOF (QTOF) system and the Triple Quad or the QTRAP systems.

ABSTRACTOur objective was to characterize lipid profiles in cell models of adipocyte differentiation in comparison to mouse adipose tissues in vivo. A novel lipid extraction strategy was combined with global lipid profiling using direct infusion and sequential precursor ion fragmentation, termed MS/MSALL. Perirenal and inguinal white adipose tissue and interscapular brown adipose tissues from adult C57BL/6J mice were analyzed. 3T3‐L1 preadipocytes, ear mesenchymal progenitor cells, and brown adipose‐derived BAT‐C1 cells were also characterized. Over 3000 unique lipid species were quantified. Principal component analysis showed that perirenal versus inguinal white adipose tissues varied in lipid composition of triacyl‐ and diacylglycerols, sphingomyelins, glycerophospholipids and, notably, cardiolipin CL 72:3. In contrast, hexosylceramides and sphingomyelins distinguished brown from white adipose. Adipocyte differentiation models showed broad differences in lipid composition among themselves, upon adipogenic differentiation, and with adipose tissues. Palmitoyl triacylglycerides predominate in 3T3‐L1 differentiation models, whereas cardiolipin CL 72:1 and SM 45:4 were abundant in brown adipose‐derived cell differentiation models, respectively. MS/MSALL data suggest new lipid biomarkers for tissue‐specific lipid contributions to adipogenesis, thus providing a foundation for using in vitro models of adipogenesis to reflect potential changes in adipose tissues in vivo. J. Cell. Biochem. 117: 2182–2193, 2016. © 2016 Wiley Periodicals, Inc.

This study was conducted to assess the value of a high resolution, high mass accuracy time-of-flight analyzer in combination with nanoliquid chromatography for the analysis of polyphenols and their metabolites. The goal was to create a method that utilizes small volumes of biological fluids and provides a significant improvement in sensitivity compared with existing methods. Accordingly, nanoLC-MS and nanoLC-pseudo-multiple reaction monitoring (MRM) methods were developed that had a lower limit of quantification of 0.5 nM for several polyphenols and were linear over 2-3 orders of magnitude (R-2 > 0.999). Using urine samples, the ability to observe and quantify polyphenols in such a complex biological fluid depended on much narrower mass windows (0.050 amu or less) on a TOF analyzer than those used on a quadrupole analyzer (0.7 amu). Although a greater selectivity was possible with the low mass resolution of a triple quadrupole instrument using the MRM approach, for the daidzein metabolite O-DMA, a chromatographically resolvable second peak could only be substantially reduced by using a 0.01 amu mass window. The advantage of a TOF analyzer for product ion data is that the whole MSMS spectrum is collected at high mass accuracy and MRM experiments are conducted in silica after the analysis. (C) 2014 Elsevier Inc. All rights reserved.

Nonalcoholic fatty liver disease (NAFLD) is associated with altered hepatic lipid composition. Animal studies suggest that the hepatic ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) contributes to steatogenesis and inflammation. This ratio may be influenced by dysregulation of the PE N-methyltransferase (PEMT) pathway or by a low-choline diet. Alterations in the liver may also influence lipid composition in circulation such as in erythrocytes, which therefore may have utility as a biomarker of hepatic disease. Currently, no study has assessed both liver and erythrocyte PC/PE ratios in NAFLD. The aim of this study was to compare the PC/PE ratio in the liver and erythrocytes of patients with simple steatosis (SS) or nonalcoholic steatohepatitis (NASH) with that of healthy controls. PC and PE were measured by mass spectrometry in 28 patients with biopsy-proven NAFLD (14 SS, 14 NASH) and 9 healthy living liver donors as controls. The hepatic PC/PE ratio was lower in SS patients (median [range]) (1.23 [0.27-3.40]) and NASH patients (1.29 [0.77-3.22]) compared with controls (3.14 [2.20-3.73]); both p < 0.001) but it was not different between SS and NASH. PC was lower and PE higher in the liver of SS patients compared with controls, whereas in NASH patients only PE was higher. The PC/PE ratio in erythrocytes was also lower in SS and NASH patients compared with controls because of lower PC in both patient groups. PE in erythrocytes was not different among the groups. In conclusion, NAFLD patients have a lower PC/PE ratio in the liver and erythrocytes than do healthy controls, which may play a role in the pathogenesis. The underlying mechanisms require further investigation.

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80–114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.

The TripleTOF 5600 System, a hybrid quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications that included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare with other MS technologies and to demonstrate the abilities of the instrument platform.

The growth in lipidomics research is uncovering a need for complete and comprehensive workflows for identifying and quantifying lipid species from biological extracts. In choosing analytical methods for lipidomics, different yet complementary mass spectrometry approaches can provide a more complete and comprehensive data set leading to a detailed characterization of lipid molecular species from complex extracts. A preliminary strategy carried out as global “shotgun” tandem mass spectrometry by direct infusion electrospray ionization or LC-MS/MS analysis, uses information dependant MS/MS scanning in both polarities for unbiased lipid profiling. The second approach involves multiple lipid-class-specific precursor ion and neutral loss scanning whose resulting spectra can be used directly to identify and characterize lipids and fatty acids in tissue-derived lipid extracts. The multiple precursor ion scanning (MPIS) methods have been published and recently reviewed by Ekroos et al, describing the advantages of targeted MPIS techniques for generating comprehensive lipid arrays from small sample volumes. Fully characterizing these lipid components by high quality MS/MS for fatty acid chain length and double bond positioning is a critical step for understanding their biological implications in cell signaling and lipid-initiated disease progression. Taking advantage of the speed, selectivity, and sensitivity of hybrid triple quadrupole technology, whole lipid extracts from rat brain tissue can be analyzed by direct nanoESI infusion for in depth glycerophospholipid profiling - achieving both qualitative and quantitative data (with the use of synthetic lipid internal standards) in very fast analysis times. Lipid species identification and quantitation is carried out using LipidView™ Software enabling post acquisition processing of precursor ion, neutral loss, MRM, and MS/MS data via lipid database searching and accurate peak integration. We present robust targeted and global workflows for the identification and quantitation of glycerphospholipids in total lipid extracts from rat brain tissue.

Qualitative mass spectrometry experiments are most commonly performed by information dependent acquisition strategies, where a survey scan is collected, and precursors are selected for MS/MS analysis based on a set of user defined criteria. This is widely applied for LC-MS/MS analyses in qualitative screening, semi-quantitative profiling, and compound identification experiments. However, for some experiments such as the analysis of complex lipid samples, information independent strategies can offer some key advantages. A simple informationindependent technique involves stepping a mass isolation window across a set mass range in small increments, fragmenting selected precursor ions in a collision cell, and recording all product ions. As previously attempted with ion trapping MS platforms in past years, MS/MS of all precursors ions is highly desirable as an un-biased profiling technique as nothing is missed and all product ion data can be mined retrospectively. Quadrupole time of flight (QqTOF) instruments are well suited to data-independent experiments as they provide high resolution and mass accuracy and without speed or mass range limitations. Furthermore, the strength of QqTOF instruments for lipid molecular identification applications has been well demonstrated. Here, we review the data independent Infusion MS/MS Workflow and its application towards the comprehensive analysis of biological lipid extracts (Figure 1).

Vasopressin controls water excretion through regulation of aquaporin-2 (AQP2) trafficking in renal collecting duct cells. Using mass spectrometry, we previously demonstrated four phosphorylated serines (Ser256, Ser261, Ser264, and Ser269) in the carboxyl-terminal tail of rat AQP2. Here, we used phospho-specific antibodies and protein mass spectrometry to investigate the roles of vasopressin and cyclic AMP in the regulation of phosphorylation at Ser269 and addressed the role of this site in AQP2 trafficking. The V2 receptor-specific vasopressin analog dDAVP increased Ser(P)269-AQP2 abundance more than 10-fold, but at a rate much slower than the corresponding increase in Ser256 phosphorylation. Vasopressin-mediated changes in phosphorylation at both sites were mimicked by cAMP addition and inhibited by protein kinase A (PKA) antagonists. In vitro kinase assays, however, demonstrated that PKA phosphorylates Ser256, but not Ser269. Phosphorylation of AQP2 at Ser269 did not occur when Ser256 was replaced by an unphosphorylatable amino acid, as seen in both S256L-AQP2 mutant mice and in Madin-Darby canine kidney cells expressing an S256A mutant, suggesting that Ser269 phosphorylation depends upon prior phosphorylation at Ser256. Immunogold electron microscopy localized Ser(P)269-AQP2 solely in the apical plasma membrane of rat collecting duct cells, in contrast to the other three phospho-forms (found in both apical plasma membrane and intracellular vesicles). Madin-Darby canine kidney cells expressing an S269D “phosphomimic” AQP2 mutant showed constitutive localization at the plasma membrane. The data support a model in which vasopressin-mediated phosphorylation of AQP2 at Ser269:(a) depends on prior PKA-mediated phosphorylation of Ser256 and (b) enhances apical plasma membrane retention of AQP2.

Large-scale identification of phosphotyrosyl (pTyr)-proteins by LC-MS methods remains challenging. We previously developed a novel isotopic labeling method, called IVICAT (In Vacuo Isotope-Coded Alkylation Technique) that improves the detection sensitivity of pTyr-containing peptides in positive ion ESI-MS by trimethylating the N-terminus of every peptide, thereby creating a fixed positive charge. Here we use pTyr immunoprecipitation, IVICAT labeling, IMAC, and LC-MS/MS to profile the pTyr proteome of inner medullary collecting duct (IMCD) cells isolated from rat kidneys. These experiments identified 114 pTyr sites, 103 of which have not been previously reported. Using H3- or D3-methylating reagents in IVICAT, we quantified pTyr changes in response to the V2 receptor selective analog dDAVP in IMCD cell suspensions. Use of our in house software (QUIL) for construction of pseudochromatograms from time-dependent LC-MS data resulted in the discovery of several sites modified by vasopressin including phospholipase C gamma-1 (PLCγ1) at Y771 (increase of approximately two-fold confirmed by immunoblotting). Differential centrifugation showed that dDAVP displaces PLCγ1 from membrane to cytosol, consistent with a net inhibitory action. The data indicate that vasopressin signaling includes changes in tyrosine phosphorylation that have potential roles in regulation of aquaporins or urea transporters.

Hypertonicity increases the activity of TonEBP, resulting in increased transcription of osmo-protective genes. Hypertonicity also increases phosphorylation of TonEBP and there is indirect evidence that phosphorylation of some specific amino acids may be involved in its activation. However, there has been no direct demonstration of phosphorylation of any specific amino acid. The goal of the present studies was to identify amino acids in TonEBP that are phosphorylated. We used HEK293 cells stably transfected with TonEBP-1-547-V5. We incubated them at 200 or 500 mosmol/kg (NaCl varied) for two hours, extracted nuclear and cytoplasmic proteins, immunoprecipitated TonEBP-V5, subjected the immunoprecipitate to in-solution digestion, enriched for phosphopeptides by immobilized metal affinity chromatography (IMAC), and analyzed peptides from both the eluate and flow-through by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In order to maximize coverage of possible phosphorylation sites, we used 3 different proteolytic enzymes (trypsin, endoproteinase Arg-C, and proteinase K). We find a high probability that several different serines, threonines and tyrosines are phosphorylated in at least one of the conditions. We are mutating the amino acids that we identified to determine their role in TonEBP activity.

We examined the association of acetyl-CoA:α-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4°C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.