Papac D I, Wong A, Jones A J
Department of Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080, USA.
Anal Chem. 1996 Sep 15;68(18):3215-23. doi: 10.1021/ac960324z.
2,5-Dihydroxybenzoic acid (DHB) is the most commonly used matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) of oligosaccharides. Because acidic, sialylated oligosaccharides are detected at only the low picomole level with DHB, alternative matrices were screened to identify a matrix with a lower limit of detection. Negative-ion spectra of pure mono-, di-, and trisialylated oligosaccharides were acquired with either 6-aza-2-thiothymine or 2',4',6'-trihydroxyacetophenone (THAP) in the linear mode. Detection limits of less than 50 fmol with signal-to-noise ratios of better than 5:1 were achieved with both matrices. THAP was the preferred matrix because it provided a lower limit of detection and gave less prompt fragmentation. Incorporation of ammonium citrate into the matrix, along with vacuum drying of the sample, was required in order to obtain maximum sensitivity with THAP. No evidence of competition for ionization was found when a mixture of mono-, di-, and trisialylated oligosaccharides was analyzed with THAP. These findings suggest that MALDI/TOF analysis may provide a rapid means to identify changes in carbohydrate composition in glycoproteins. In addition, THAP offered improved sensitivity for detection of acidic glycopeptides over alpha-cyano-4-hydroxycinnamic acid.
2,5-二羟基苯甲酸（DHB）是寡糖基质辅助激光解吸/电离飞行时间质谱（MALDI/TOF）中最常用的基质。由于使用DHB时，酸性、唾液酸化寡糖仅在低皮摩尔水平被检测到，因此筛选了替代基质以确定具有更低检测限的基质。使用6-氮杂-2-硫胸腺嘧啶或2',4',6'-三羟基苯乙酮（THAP）以线性模式采集了纯单唾液酸、双唾液酸和三唾液酸寡糖的负离子光谱。两种基质均实现了检测限低于50飞摩尔且信噪比优于5:1。THAP是首选基质，因为它提供了更低的检测限且产生的即时碎片化较少。为了使用THAP获得最大灵敏度，需要将柠檬酸铵加入基质中，并对样品进行真空干燥。当用THAP分析单唾液酸、双唾液酸和三唾液酸寡糖的混合物时，未发现电离竞争的证据。这些发现表明，MALDI/TOF分析可能提供一种快速方法来鉴定糖蛋白中碳水化合物组成的变化。此外，与α-氰基-4-羟基肉桂酸相比，THAP对酸性糖肽的检测灵敏度更高。
