The effect of freeze/thaw cycles on reproducibility of metabolic profiling of marine microalgal extracts using direct infusion high-resolution mass spectrometry (HR-MS).

Hans Chr Eilertsen,Maria Degerlund,Siv Huseby,Richard A Ingebrigtsen,Gunilla K Eriksen,Espen Hansen

doi:10.3390/molecules191016373

Abstract

During normal sample preparation, storage in freezers and subsequent freeze/thaw cycles are commonly introduced. The effect of freeze/thaw cycles on the metabolic profiling of microalgal extracts using HR-MS was investigated. Methanolic extracts of monocultures of Arctic marine diatoms were analyzed immediately after extraction, after seven days of storage at −78 °C (one freeze/thaw cycle), and after additional seven days at −20 °C (two freeze/thaw cycles). Repeated direct infusion high-resolution mass spectrometry analysis of microalgae extracts of the same sample showed that reproducibility was ca. 90% when a fresh (unfrozen) sample was analyzed. The overall reproducibility decreased further by ca. 10% after the first freeze/thaw-cycle, and after one more freeze/thaw cycle the reproducibility decreased further by ca. 7%. The decrease in reproducibility after freeze-thaw cycles could be attributed to sample degradation and not to instrument variability.

Highlights

Over the last two decades the use of mass spectrometry (MS) for quantitative and qualitative analysis of biological molecules has been increasingly applied in different fields of biological research, in medical biology and pharmacology and in ecology and taxonomy [1,2].MS has become an important analytical tool in many biological labs
If a compound in a sample was chemically altered during the experiment there would be a mass change and it would appear as a different m/z signal, decreasing the number of common signals between the two analyses
Our results indicate that a maximum of 90% reproducibility can be achieved at 150 ppm accuracy when analyzing fresh extracts using direct infusion HR-MS

Summary

Introduction

Over the last two decades the use of mass spectrometry (MS) for quantitative and qualitative analysis of biological molecules has been increasingly applied in different fields of biological research, in medical biology and pharmacology and in ecology and taxonomy [1,2]. Improvements in mass filters and ion optics have made it possible to acquire accurate mass data at high resolution with relatively affordable bench top instruments [3] This has greatly facilitated the identification of unknown compounds [4]. When performing metabolic fingerprinting based on HR-MS data two different strategies are possible, i.e., either a direct infusion of the sample into the MS or a chromatographic separation of the compounds prior to analysis in the MS [8]. Both gas and liquid chromatography (GC and LC) can be used for metabolic profiling [9]. In order to test the reproducibility of our direct infusion HR-MS method prior to and after freezing, we performed repeated analyses on extracts from the same diatom sample

Results and Discussion

Sample Preparation

HR-MS Analysis

Data Processing

Conclusions