Charles “Chuck” Sweeley Jr., who made major contributions to the fields of sphingolipids and mass spectrometry, died on Sept. 21 in Lansing, Mich., after a long battle with a rare form of bladder cancer. He was 82.
A native of Williamsport, Pa., Chuck earned a bachelor’s degree in chemistry at the University of Pennsylvania in 1952 and a Ph.D. in biochemistry at the University of Illinois, Urbana-Champaign, in 1955, working under the direction of Herbert Carter. After training with Evan Horning at the National Institutes of Health, Chuck took a position at the University of Pittsburgh in 1960 and was promoted to professor in 1966. He moved to Michigan State University in 1968, served as chairman of the biochemistry department from 1979 to 1985 and retired in 1992.
Here we highlight some of his accomplishments, which are described in greater detail in a recent review (1).
Chuck’s illustrious research career began during his Ph.D. training. His thesis was on the chemistry of antibiotics, one of the major interests of the Carter laboratory. However, he spent most of his career studying the chemistry and biochemistry of sphingolipids and glycosphingolipids, the other major focus of the Carter laboratory (1).
Chuck was the first to develop a sensitive method for determining the sphingoid bases using periodate oxidation and analysis of the resultant long-chain fatty aldehydes by gas-liquid chromatography, or GLC, then a novel technology that Chuck played a key role in developing. Chuck wrote:
“An unexpected, career-altering opportunity came to me when Horning ordered the first gas chromatograph at the National Institutes of Health and I was given the task of setting up this machine … Later, I set out independently to apply gas-liquid chromatography … to other lipids. We reported a new method to analyze sphingolipid bases in sphingomyelin and glycosphingolipids by conversion of these long-chain bases to aldehydes with periodate and separation by GC … Human plasma sphingomyelin was found to contain sphingosine, dihydrosphingosine, and two unknown bases which were later shown to be sphinga-4,14-dienine and hexadecasphing-4-enine”(1).
His method to hydrolyze sphingolipids is still used today to analyze the de novo biosynthesis of sphingolipids by following labeling of the sphingoid base backbone or to quantify sphingolipids on the basis of the amount of the sphingoid bases released. Chuck was the first to characterize a novel unsaturated sphingoid base, sphinga-4,14-dienine, in the sphingomyelin fraction of human plasma.
With the characterization of sphingoid bases as a background, he further investigated the biosynthesis of sphingosine as a condensation product of palmitoyl Co-A and serine, and, employing elegant biochemical tools, he elucidated the stereochemistry of the reaction intermediates and products. These elegant studies led to a proposed mechanism for how the first sphingolipid intermediate, 3-ketosphinganine, is formed by removal of the α-proton from serine as the Schiff base with pyridoxal 5-phosphate, displacement of the coenzyme A moiety from palmitoyl-CoA to form the carbon–carbon bond and then decarboxylation. This mechanism has been borne out by subsequent spectroscopic and X-ray crystallographic studies. His lab also demonstrated that double bonds in the sphingoid base and the 4-hydroxyl group of phytosphingosine are added after 3-ketosphinganine has been made.
Chuck was also the first to introduce derivatization of complex and simple sugars by the trimethylsilyl, or TMS, hydroxyl protecting group. This chemical maneuver rendered these and related compounds sufficiently volatile and thermally stable that they could pass through a gas chromatograph. Conversion to TMS derivatives greatly facilitated analysis of nonvolatile compounds owing to the ease in sample preparation and predicable elution profiles. Previously, these natural products were converted to volatile peracetyl or permethyl derivatives for GLC analysis. Chuck’s 1963 paper on TMS derivatization of carbohydrates (2) was one of the 500 most-cited papers of the 1960s. The success of his strategy to derivatize sugars also was made possible by his introduction of the stationary phase, SE-30, for GLC.
With Dennis Vance, then one of his doctoral students, Chuck was one of the early investigators to utilize stable isotopes, especially deuterium, to assist in elucidating the metabolism of glycosphingolipids and carbohydrates.
Chuck first recognized Fabry’s disease as a lysosomal glycolipid-storage disease and went on to isolate and partially characterize one of the major accumulated glycosphingolipids as trihexosyl globoside. He wrote of his good fortune in meeting Bernard Klionsky, who told him “about a rare genetic disorder called Fabry’s disease, supposedly a sphingomyelin disorder.” Chuck wrote, “I was pleased that he was willing to give me a piece of formalin-fixed kidney from a Fabry patient … It did not take long to find that this kidney contained abnormal amounts of two novel glycosphingolipids” (1).
Although the glycosidic linkage of the terminal galactosyl residue was wrongly assigned the β configuration, Chuck always acknowledged at scientific meetings and in publications the contributions from other investigators who showed it was actually of the α configuration — a true reflection of his graciousness and generosity. His work on the lysosomal glycosphingolipid-storage disorders led to the characterization of many serum neutral glycosphingolipids and to the study of a variety of glycosidases in animal and plant sources. These studies provided insights into the nature of lysosomal glycolipid-storage disorders and paved the way for the development of enzyme-replacement therapy for lysosomal lipid-storage disorders.
Chuck undertook an investigation of the biosynthesis of gangliosides, in particular GM3, or hematoside, and purified a sialyltransferase from rat liver to homogeneity employing classical biochemical techniques and affinity column chromatography. This was a remarkable feat, as glycosyltransferases in general are of very low abundance in tissue. He further elucidated the biological function of the interconversion of GM3 and lactosylceramide in human fibroblasts in relation to cellular proliferation.
He made important contributions to the emerging technique of biochemical MS in terms of analytical instrumentation, applications to the analysis of complex lipids and the use of stable isotope-labeled precursors as a strategy to study lipid biochemistry. By the late 1960s, he was using combined GC-MS in the studies of sphingolipid bases and publishing about the extraordinary power of this approach. His interest in using stable isotope labeling in biochemical studies directly led him to observe a problem caused by a separation of deuterium-labeled molecules from the corresponding protium species by GS. This feature, resulting from an isotope effect, complicated analysis of the isotope ratios of peaks eluting from the gas chromatograph. At this time, Chuck was on sabbatical in Ragnar Ryhage’s laboratory at the Karolinska Institute, and Ryhage’s lab was developing one of the first GC-MS instruments, the LKB 9000. To address the isotope-effect problem, a method was developed to switch the ion source acceleration potential in a rapid fashion to focus alternatively the appropriate isotope-labeled ions at the detector, thus enabling specific ions to be sampled rapidly at an appropriate time scale for elution from the gas chromatograph. This voltage-alternation approach was published in 1966, and the concept of selected ion recording remains a mainstay of GC-MS and LC-MS techniques.
Sweeley was before his time in promoting the power of time-of-flight MS, or TOF-MS. Using a fast TOF detector, he showed that it was possible to obtain 10 complete mass spectra per second during a GC separation of extracts of biological fluids using a time array detection strategy. While the true speed potential for TOF-MS would have to wait for the development of fast-timing circuits and faster data-acquisition systems, he used this concept of rapid mass-to-charge scanning to reveal the wealth of molecules present in urine and other biological fluids, a type of study he called metabolic profiling and a prototype for what we know now as metabolomics. Indeed, his metabolic profiling was decades ahead of its time. Chuck described it this way:
“Our first paper was on the development of an on-line computer system for single focusing mass spectrometry (1970). This was followed by a report on computer-controlled multiple ion detection in combined gas chromatography-mass spectrometry (GC-MS) (1973) and development of a computer system for selected ion monitoring of multi-component mixtures by computer control of accelerating voltage and magnetic field strength (1975). This allowed investigators to determine several substances in mixtures at the very high sensitivity obtained by selected ion monitoring. The next step was to develop methods for the automated determination of many substances in a mixture, and this led to the development of MSSMET, a computer system for metabolic profiling (1974 – 1986). We utilized metabolic profiling to examine the urinary organic acid fraction in natural early-onset insulin-dependent diabetic dogs (1988) and in studies of the turnover of [U-14C]-glucose into various metabolites in lactic acidemias (1988). This technique was utilized not only in studies of urinary organic acids but also in the analysis of urinary steroids … Metabolic profiling was also extended to a new and novel detection system using musical sounds instead of graphs or tables to analyze normal and abnormal samples of urine (1987). Intensities at the apex of each GC peak were converted to frequencies and played on a digital keyboard, higher notes reflecting greater concentrations of metabolites. This was one of the first reports on the use of sound as a sense of perception in the field of analytical chemistry and became known whimsically in the press world-wide as ‘musical urines.’” (1)
In closing comments, Chuck noted:
“By now the work I have described is ancient history … But I lived in exciting times, times that marked the beginnings in most of the areas of my research. It was the beginning of gas chromatography, nearly the beginning of mass spectrometry in the biomedical sciences, the beginning of chemistry and metabolism of sphingolipids, and certainly the beginning of what we now know about intermediary metabolism in man. Our generation provided a foundation upon which modern investigation in these fields has grown and prospered.” (1)
- 1. Sweeley, C. Jpn. Acad. Ser. B Phys. Biol. Sci. 86, 822 – 836 (2010).
- 2. Sweeley, C. et al. J. Am. Chem. Soc. 85, 2497 – 2507 (1963).