Ruben Yiqi Luo, PhD, DABCC, Assistant Professor, Department of Pathology, School of Medicine, Stanford University
Cerebrospinal fluid (CSF) leak may lead to potentially life-threatening meningitis if left untreated. β2-transferrin (β2-Tf) is a proteoform of human transferrin (Tf) mainly present in CSF and barely detectable in other body fluids. Therefore, β2-Tf in secretion samples can be used as a marker to diagnose CSF leak. The clinical utility and diagnostic value of β2-Tf in CSF leak have been demonstrated. β2-Tf, together with the typical Tf proteoform in serum, β1-Tf, were named after their electrophoretic mobility in gel electrophoresis. However, the structures of β1-Tf and β2-Tf have not been elucidated. Dr. Ruben Luo’s research team used a novel affinity capture sample preparation technique, microprobe-capture in-emitter elution (MPIE), combined with mass spectrometry (MS) to study the structures of β1-Tf and β2-Tf.
As a sample preparation technique, MPIE utilizes the analytical power of a label-free optical sensing technology (next-generation biolayer interferometry, BLI) to overcome the challenge of lack of process monitoring in the conventional affinity capture-MS analysis. To implement MPIE, an analyte is first captured on the surface of a microprobe and subsequently eluted from the microprobe inside an electrospray emitter. The analyte is immediately ionized via electrospray ionization (ESI) for MS analysis, achieving the direct coupling between MPIE and MS. The capture process is monitored in real-time via BLI. By this means, BLI and MS are combined in the form of MPIE-ESI-MS, which is readily deployed to analyze the Tf glycoforms and elucidate the structures of β1-Tf and β2-Tf. The experiment workflow of MPIE-ESI-MS for Tf analysis is shown in Figure 1.
Based on the MPIE-ESI-MS results of serum, CSF, and secretion samples, the structures of β1-Tf and β2-Tf were elucidated. As Tf glycoforms, β1-Tf and β2-Tf share the amino acid sequence and intramolecular disulfide bonds but have varying N-glycans. β1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611, while β2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611, as shown in Figure 2.
A set of 11 secretion samples from patients suspected of CSF leak were analyzed using the MPIE-ESI-MS method, among which 5 samples were positive for β2-Tf, and the rest were negative as measured by IFE test and clinical manifestations. The MS peak at 78008 Da was observed in the MPIE-ESI-MS results of the 5 positive samples but not found in those of the 6 negative samples, which confirmed the consistency between the MPIE-ESI-MS method and the conventional immunofixation electrophoresis test.
Figure 1. Experiment workflow of MPIE-ESI-MS for Tf analysis. (A) Running BLI-based affinity capture. (B) Setting up the in-emitter elution ESI-MS with pictures of a BLI microprobe, an electrospray emitter, and an MPIE-ESI-MS interface.
Figure 2. (A) Primary structure of human transferrin, showing the sequence of 679 amino acids, 19 disulfide bonds, and 2 N-glycosylation sites. (B) N-glycan structures on β1-Tf (major serum-type Tf) and β2-Tf (major brain-type Tf), confirmed by comparing the theoretical molecular masses of the Tf glycoforms with the measured molecular masses.
使用微探针捕获喷针内洗脱和高分辨质谱研究 β1-转铁蛋白和 β2-转铁蛋白
脑脊液外漏是一种严重的临床症状，如果不及时治疗可能会危及生命。 β2-转铁蛋白（β2-Tf）是人转铁蛋白(Tf)的一种蛋白质形态，主要存在于脑脊液中，在其他体液中几乎检测不到。因此，分泌物样本中的β2-Tf可以作为脑脊液外漏的诊断标记物。β2-Tf 在脑脊液外漏中的临床实用性和诊断价值已得到证实。 β2-Tf 与血清中典型的 Tf 蛋白质形态 β1-Tf 因其在凝胶电泳中的条带区域得名。 然而，β1-Tf和β2-Tf的结构尚未阐明。Dr. Ruben Luo的研究组使用一种新型亲和捕获样品制备技术——微探针捕获喷针内洗脱（MPIE）与质谱相结合来研究β1-Tf和β2-Tf的结构。
作为一种样品制备技术，MPIE 利用非标记光学传感技术（生物膜干涉测量法，BLI）的分析能力来克服传统亲和捕获质谱分析中缺乏过程监控的挑战。 为了实施 MPIE，分析物首先被亲和捕获在微探针的表面上，然后在电喷雾喷针内洗脱，通过电喷雾电离 (ESI) 立即将分析物离子化进行质谱分析，实现 MPIE 与质谱之间的直接耦合。分析物的亲和捕获过程可以通过 BLI 进行实时监测。通过这种方式，BLI 和质谱以 MPIE-ESI-MS 的形式组合，可以用于分析 Tf 蛋白质形态并阐明 β1-Tf 和 β2-Tf 的结构。使用MPIE-ESI-MS进行Tf分析的实验流程如图1所示。
根据血清、脑脊液和分泌物样本的MPIE-ESI-MS分析结果， β1-Tf和β2-Tf的结构得以阐明。 作为具备不同糖型的Tf蛋白质形态，β1-Tf和β2-Tf共享氨基酸序列和分子内二硫键，但是带有不同的N-聚糖。 β1-Tf是主要血清型Tf，在Asn413和Asn611上有两个G2S2 N-聚糖，而β2-Tf是主要脑脊液型Tf，在Asn413上有一个M5 N-聚糖，在Asn611上有一个G0FB N-聚糖 ，如图2所示。
采用 MPIE-ESI-MS 方法对疑似脑脊液外漏患者的 11 份分泌物样本进行分析，其中 5 份样本经 IFE 检测和临床表现检测呈 β2-Tf 阳性，其余样本呈阴性。 5个阳性样本的MPIE-ESI-MS分析结果中观察到了78008 Da的质谱峰，而6个阴性样本的MPIE-ESI-MS分析结果中未发现该质谱峰，验证了MPIE-ESI-MS方法与传统的免疫固定电泳方法之间的一致性。
Reference
[1] Luo RY, Yang S. Microprobe-Capture In-Emitter Elution: An Affinity Capture Technique to Directly Couple a Label-Free Optical Sensing Technology with Mass Spectrometry for Protein Analysis. Anal Chem 95:5494-5499, 2023.
[2] Luo RY, Pfaffroth C, Yang S, Hoang K, Yeung PSW, Zehnder JL, Shi RZ, “Study of β1-Transferrin and β2-Transferrin Using Microprobe-Capture in-Emitter Elution and High-Resolution Mass Spectrometry”, Sci Rep 13:14974, 2023.