An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Summary

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Abstract

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

Introduction

Carbohydrate analysis is an important and challenging subject. Carbohydrates and their derivatives play important roles in living organisms^1,2,3. These molecules have complicated structures and are prone to decompose. Many of them cannot be clearly characterized due to difficulties in separation and detection. Although matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been applied to analysis of a wide range of biomolecules, due to its sensitivity and comprehensible results⁴, analyzing carbohydrates using MALDI-MS continues....

Protocol

1. Sample Plate Preconditioning

1. Cleaning of the sample plate
   1. Wear nitrile gloves to avoid contamination of the sample plate during cleaning.
   2. Hand-wash the sample plate with 100.0 mL of detergent solution (1.0 mg/mL).
   3. Hand-wash the sample plate with distilled-deionized water (DDW).
   4. Rinse the sample plate surface with 30.0 mL of MeOH.
   5. Put the sample plate in a 600 mL beaker and fill with DDW until the sample plate is fully immersed in water.
   6. Put the beaker in an ultrasonic bath (see Table of Materials) and sonicate the sample plate for 15 min....

Results

Representative SEM images of SLe^A mixed with DHB prepared using DD and recrystallization methods are shown in Figure 1. A typical DHB morphology as prepared by the DD method is large needle-shaped crystals at the rim and fine crystalline structures in the center of sample spots. The typical lengths of such needle-shaped crystals are ~100 µm. After recrystallization by MeOH, the sample has a larger area covered evenly with fine flake-like crystals. .......

Discussion

Sample heterogeneity is a crucial problem in MALDI-MS. DD is the most commonly used sample preparation method, but the resultant crystals are highly heterogeneous. Such samples show poor shot-to-shot and sample-to-sample signal reproducibility. Therefore, searching for "sweet spots" in sample areas during data acquisition is a common procedure in MALDI experiments. Such heterogeneous samples are unsuitable for quantification in routine analyses.

In the current study, MALDI sample morph.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
Detergent powder	Alconox	242985
Methanol	Merck	106009
Acetonitrile	Merck	100003
2,5-dihydroxybenzoic acid (DHB)	Alfa Aesar	A11459
sialyl-lewis A (SLeA)	Sigma-Aldrich	S1782
Maltoheptaose	Sigma-Aldrich	M7753
Pipette tips	Mettler Toledo	17005091
Microcentrifuge tube	Axygen	MCT-150-C
Equipment
Milli-Q water purification system	Millipore	ZMQS6VFT1
Powder-free nitrile gloves	Microflex	SU-690
600 mL beaker	Duran	2110648
Ultrasonic cleaner	Delta	DC300H
Hygrometer	Wisewind	5330
Nitrogen gas flowmeter	Dwyer	RMA-6-SSV
K-type thermocouples	Digitron	311-1670
Vortex mixer	Scientific Industries	SI-0236
Mini centrifuge	Select BioProducts	Force Mini
Pipette	Rainin	pipet-lite XLS
Stereomicroscope	Olympus	SZX16
Temperature controllable drying chamber	This lab
Ultraflex II TOF/TOF mass spectrometer	Bruker Daltonics
MTP 384 target plate polished steel BC	Bruker Daltonics	8280781
Flexcontrol Version 3.4	Bruker Daltonics		Control software
Fleximaging Version 2.1	Bruker Daltonics		Imaging software
Flexanalysis Version 3.4	Bruker Daltonics		Analysis software