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Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is increasingly recognized for its potential in the discovery of novel biomarkers directly from tissue sections. However, there are no MALDI IMS studies as yet on the adipose tissue, a lipid-enriched tissue that plays a pivotal role in the development of obesity-associated disorders. Herein, we aimed at developing an optimized method for analyzing adipose tissue lipid composition under both physiological and pathological conditions by MALDI IMS. Our studies showed an exacerbated lipid delocalization from adipose tissue sections when conventional strategies were applied. However, our optimized method using conductive-tape sampling and 2,5-dihydroxybenzoic acid (DHB) as a matrix, preserved the anatomical organization and minimized lipid diffusion from sample sections. This method enabled the identification of a total of 625 down-regulated and 328 up-regulated m/z values in the adipose tissue from a rat model of extreme obesity as compared to lean animals. Combination of MALDI IMS and liquid chromatography (LC)-MS/MS data identified 44 differentially expressed lipid species between lean and obese animals, including phospholipids and sphingomyelins. Among the lipids identified, SM(d18:0_18:2), PE(P-16:0_20:0), and PC(O-16:0_16:1) showed a differential spatial distribution in the adipose tissue of lean vs. obese animals. In sum, our method provides a valuable new tool for research on adipose tissue that may pave the way for the identification of novel biomarkers of obesity and metabolic disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In these experiments we have studied the in vitro metabolism of LVV-hemorphin-7 in human plasma by using reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with micro-electrospray mass spectrometry (micro-ES-MS). Tandem mass spectrometry (MS-MS) was performed in order to verify the structure of the peptide fragments found. Incubations were performed with and without different protease inhibitors. Results showed that LVV-hemorphin-7 was metabolized from the N-terminal end of the peptide, probably by an amastatin-sensitive exopeptidase.

The application of reversed-phase HPLC in combination with micro-electrospray mass spectrometry to study a substance P (SP)-hydrolysing endoprotease in human cerebrospinal fluid (hCSF) is reported. The enzyme was partially purified from the hCSF specimens by ion-exchange chromatography and molecular sieving. During the purification procedure the enzyme activity was monitored by measuring the formation of the SP-fragment 1-7 from SP by radioimmunoassay. Regarding its behaviour upon molecular sieve chromatography, the enzyme was suggested to be associated with an apparent molecular mass of around 100 x 10(3). In subsequent experiments using the partially purified endopeptidase, the hydrolysis of SP was demonstrated by HPLC. The reaction product mixture was resolved in several components including the N-terminal fragments 1-8, 1-7 and 1-6 and the C-terminal fragment 8-11. The identity of these fragments were confirmed by tandem mass spectrometry. It was concluded that the present SP-degrading enzyme is different from those previously identified and purified from hCSF. The applied techniques were proven to be highly efficient for the recovery and identification of the released peptide products.

The utility of the combination of separations techniques, such as liquid chromatography and capillary zone electrophoresis, with mass spectrometry in applications involving protein analysis is discussed. The use of continuous-flow fast atom bombardment and electrospray ionization mass spectrometry is compared for the analysis of tryptic digests. For liquid chromatography, both microbore and slurry-packed capillary bore columns were used to separate peptides from proteolytic digests.

Reversed-phase high-performance liquid chromatography using aliphatic carboxylic acids as surfactants was used to separate basic, acidic and neutral dipeptides. The enhancement of the capacity factors of basic dipeptides versus the chain length of carboxylic acid followed sigmoidal curves. The capacity factor of dipeptides fell precipitously with addition of salts until it decreased to a value which was unaffected by the addition of more salts. For the identification of dipeptides produced by the hydrolysis of proteases (dipeptidylaminopeptidases), gas chromatographic-mass spectrometric techniques were used for acidic and neutral dipeptides. Basic dipeptides could be positively identified either by normal chromatographic procedures or by collecting the suspected peaks for further characterization by alternate techniques.

Fast atom bombardment (FAB) mass spectrometry was employed to identify and quantitate dansyl amino acids obtained in the N-terminal analysis of proteins. FAB mass spectra of dansyl amino acids are characterized by intense quasimolecular ions formed by cationization of the molecular species and fragment ions produced by cleavage of the bonds on either side of the sulfanyl group. Investigation of the dansyl amino acid responses using dansyl aminobutyric acid as an internal standard showed that dansyl amino acids can be determined quantitatively at a level of 0.1 nmol. N-terminal residue analysis was performed on a number of proteins to substantiate the technique including bovine serum albumin, pepsinogen, trypsinogen, bromelain, ribonuclease A, and bacteriophage P-22 tail protein.

Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are characterized by abundant ions formed by attachment of a proton, [M+H]+, or of an alkali ion, [M+alkali]+, to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to obtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt.

Combined capillary zone electrophoresis (CZE)-continuous-flow fast atom bombardment (CF-FAB) mass spectrometry is described for the analysis of mixtures of peptides. A 90 cm x 50 microns I.D. fused-silica capillary column was used for electrophoretic separations and was connected to the CF-FAB probe via an interface which allows a total flow into the mass spectrometer of about 5 microliters/min. Solutions of peptides were pneumatically loaded onto the CZE capillary, providing sample amounts of 0.1-20 pmol. The magnetic mass spectrometer was scanned over the desired mass range, usually between m/z 500 and 2500. Results are shown for separation and analysis of mixtures of synthetic peptides and also for protease digests of recombinant human growth hormone and horse heart cytochrome c.

The high sensitivity analysis of peptides by fast atom bombardment mass spectrometry has been achieved using a continuous flow probe for the introduction of samples contained in aqueous solutions. Small aliquots of sample (0.5-1 microliter) are injected into a solvent flow containing water/glycerol (8:2). For several peptide samples tested whose molecular weights were between 1000 and 1500 daltons, molecular ions were selectively monitored at the 20 fmol level with a signal-to-chemical noise of about 4:1, and full scan data were obtained on about 5 pmol of sample. The increase in sensitivity observed using the continuous flow probe is shown to be a consequence of both the decrease in the chemical background noise and to the increase in ion yields obtained from the bombardment of aqueous sample solutions.

XRF has broad applications in the quantitative analysis of trace elements in biological materials. Evaluation of its potential for the analysis of trace elements in human milk was performed. Precision of the method was demonstrated by the low (less than 3%) coefficient of variation in reproducibility of analyses performed within a given day, on different days, and by different operators. Standard curves were constructed for human milk and correlation was high (r greater than 0.98) for each of nine metals analyzed. The lower limits of determination and minimum detection limits were calculated for nine elements. Excellent correlation (r greater than 0.99) was achieved in comparison of XRF TO AA in analysis of zinc in human milk. Preliminary results of the analysis of milk zinc, copper, and iron throughout the initial 8 months of lactation disclose a decrease in trace metal concentration, most notable for zinc, during the first 60 days.

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.

GC-mass spectrometry was used to measure extremely low levels of fentanyl in dog plasma. Deuterated fentanyl was synthesized for use as an internal standard. Fentanyl was hydrolyzed to despropionyl fentanyl by 20% DCl in in deuterium oxide. Mass spectrometric analysis of the product revealed that the molecular ion was three mass units higher than that of the authentic despropionyl fentanyl, indicating that the deuterium exchange reactions occurred at this stage. Deuterated despropionyl fentanyl was reesterified by propionyl chloride to fentanyl-d3. The drug was assayed in biological fluids by extraction into ethyl acetate followed by analysis with GC-chemical-ionization mass spectrometry. The lowest measurable plasma fentanyl level is 500 pg/ml. The method is highly selective and is suitable for monitoring the time course of plasma drug levels. Evaluation of pharmacokinetic data from experiments using nine dogs revealed a triphasic phenomenon. No measurable amounts of the major metabolites, depropionyl fentanyl and norfentanyl, were detected.

Fast atom bombardment mass spectrometry is used for the analysis of the series of molecular products formed by the cleavage of polypeptide substrates with the exopeptidases carboxypeptidase Y and leucine aminopeptidase. By following the polypeptide molecular species rather than the released residues, sequence information is obtained regardless of the relative rates of cleavage of peptide bonds. In addition, unambiguous assignments of sequence can be made in the presence of multiple identical residues. The lower level of sensitivity for the analysis is in the picomole range. When carboxypeptidase Y is used, the method provides a specific and sensitive method for the sequencing of polypeptides from the C-terminus.

Microbore high-performance liquid chromatographic (HPLC) techniques have been combined with fast atom bombardment mass spectrometry (FAB-MS) for the mass-specific detection of mixtures of peptides produced by proteolytic hydrolysis of proteins. The continuous-flow FAB interface has been utilized for direct coupling of the microbore HPLC system and the mass spectrometer. Conditions are reported for the effective separation of 100 pmol of peptides at flow-rates of 5 microliters/min with acetonitrile gradients and 1-mm I.D. C8 columns. A comparison is also made between columns of 5 and 25 cm lengths for the separation of peptide mixtures. Data are presented for the separation of peptides on a slurry-packed C18 fused-silica capillary column with the continuous-flow HPLC-FAB-MS interface at flow-rates of 3 microliters/min.

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.