Gaucher’s Disease – A glycolipid storage disorder characterized by the accumulation of glucosylceramide in the spleen, liver, lungs, bone marrow and brain The disease is caused by a deficiency of lysosomal acid beta- glucosidase, which hydrolyzes glucosylceramide to ceramide and glucose Ceramides are the biological building blocks of Sphingolipids Introduction

Introduction cont. Sphingolipids play important roles in signal transduction Sphingolipids differ from phospholipids in being based on a lipophilic amino alcohol (sphingosine) rather than glycerol. Sphingolipids – are essential components of the plasma membrane of eukaryotic cells.

Topic Application of delayed extraction–matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in pericardial fluid, peritoneal fluid and serum from Gaucher disease patients Takehisa Fujiwaki,, a, Seiji Yamaguchi a, Masaru Tasaka a, Nobuo Sakura b and Tamotsu Taketomi c a b c a Department of Pediatrics, Shimane Medical University, 89-1 Enya-cho, Izumo , Japan b Department of Pediatrics, Hiroshima University School of Medicine, Kasumi, Minami-ku, Hiroshima , Japan c Department of Biochemistry, Research Center on Aging and Adaptation, Shinshu University School of Medicine, Asahi-machi, Matsumoto , Japan

So, what’s the point???? The point is……Delayed Extraction – Matrix Assisted Laser Desorption Ionization - Time Of Flight – Mass Spectrometry can be used to evaluate patients with Gaucher’s disease using small amounts of body fluids.

The Basics…..Matrix Assisted Laser Desorption Ionization – Time Of Flight (MALDI-TOF)

The basics cont……What is the Matrix??????? The matrix is an organic acid that holds the sample Absorbs photon energy and transfers it Reduces intermolecular forces and aggregation by serving as a solvent

The basics cont…Laser Desorption Ionization??? UV laser is used to generate ions The matrix absorbs the energy and transfers the energy to the sample The sample becomes ionized into a gas phase (proton transfer)

The basics cont……Time of Flight???? They are accelerated by high voltage and separated based on the time it takes them to travel to the detector All ions gain same kinetic energy Ions enter the mass spectrometer or time-of flight (TOF) A spectrum of ion intensity as a function of travel time is recorded The ion’s passage through the drift tube can be described as: qE = 1/2 m v 2 = 1/2 m (l/t) q= ion charge v= velocity through drift tube l= length of tubet= time of flight through tube m= ion massE= electric field voltage Time of flight can be converted to molecular mass: t ~ (m/q) 1/2

Methods Sphingolipid fractions from body fluids and serum were collected Each sample was mixed with a matrix composed of 2,5 dihydroxybenzoic acid (DHB). Advantages of DHB: ions undergo little metastable decay insensitive to contaminations produces minimal interference in the low molecular weight range Samples were loaded into a Voyager DE-RP (2.0 m flight length, reflector mode) and mass spectra of the samples was obtained in the positive ion mode with an N2 laser (337nm) and a scan average of 256. Two point external calibration was performed each time.

Delayed Extraction? Basically – it groups ions and provides better resolution A time delay between ionization of the sample and extraction from the ion source Allows ions with identical mass/charge values to arrive at the detector at the same time

External Calibration? Internal and external calibration External involves acquisition from two samples 1 st sample – two standards derive calibration equation 2 nd sample – contains unknowns and is calibrated with standards Advantage of External calibration – no risk of concentration/dynamic range or ionization suppression

Reflector mode? This results in focusing the ion packets in space and time at the detector. Basically – it gives you better resolution Ion reflector – slows down incoming ions and reverses flight path to detector

Table 1. Measured mass-to-charge ratios (m/z), and proposed molecular species associated with sphingolipids *Ceramide monohexoside includes glucosylceramide. "d" indicates dihydroxy-sphingosine.

Conclusions Control patients produce sufficient lysosomal acid B- glucosidase and can properly hydrolyze CMH to ceramide, which is a sphingomyelin precursor. Normal ceramide monohexoside/sphingomyelin (CMH/SM) peak intensity ratios circled in red. Lysosomal acid B-glucosidase Ceramide monohexoside Ceramide Glucose Sphingomyelin HYDROLYSIS

Conclusions Ceramide monohexoside/sphingomyelin (CMH/SM) peak intensity ratios are increased in different body fluids, circled in blue. Lysosamal acid B-glucosidase Ceramide monohexoside Ceramide Glucose Sphingomyelin Patients with Gaucher’s disease are deficient in lysosomal acid B- glucosidase and therefore cannot properly hydrolyze CMH to ceramide, resulting in less sphingomyelin. HYDROLYSIS