Quantitative plasma proteome of mild cognitive impairment

Multiple Affinity Removal Spin Cartridge Human 14 kit (Agilent) was used to remove 14 major proteins from plasma. Samples were denatured with an equivalent volume of trifluoroethanol and reduced with dithiothreitol. Free cysteine residues were alkylated with iodoacetamide. Samples were incubated with trypsin and desalted with C18 ZipTip. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to an TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution configuration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. A rolling collision energy (CE) parameters script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identification, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Homo sapiens, (vi) ID focus: biological modifications, (vii) detected protein threshold: > 0.05 (10% confidence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve fitting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identifications. Positive identifications were considered when identified proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH files were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

本实验采用多重亲和去除离心柱式人血浆14种蛋白去除试剂盒（Multiple Affinity Removal Spin Cartridge Human 14 kit，安捷伦）从血浆中去除14种主要蛋白。样品采用等体积三氟乙醇（trifluoroethanol）变性，并以二硫苏糖醇（dithiothreitol）还原；游离半胱氨酸残基采用碘乙酰胺（iodoacetamide）进行烷基化修饰。随后将样品与胰蛋白酶（trypsin）孵育酶解，再通过C18 ZipTip吸头完成脱盐处理。脱盐后的样品用0.1%甲酸（formic acid，FA）复溶，采用纳流液相色谱Eksigent 400系统（nanoLC Eksigent 400 system，Eksigent、AB Sciex）联用TripleTOF 6600质谱仪（TripleTOF6600 mass spectrometer，AB Sciex）进行LC-MS分析。
肽段分离采用捕集-洗脱构型的液相色谱系统，使用纳米捕集柱（350 μm × 0.5 mm，3 μm，120 Å，AB Sciex）与纳米ChromXP C18反相色谱柱（75 μm × 15 cm，3 μm，120 Å，AB Sciex），流速设置为300 nl/min，洗脱梯度如下：先以90 min线性梯度将流动相中乙腈（acetonitrile）比例从8%升至30%（流动相为0.1% FA），随后以10 min线性梯度将乙腈比例从30%升至40%（流动相为0.1% FA）。
质谱仪采用信息依赖采集（information-dependent acquisition，IDA）模式：先以250 ms扫描质量范围400–1500 m/z的全谱，随后最多进行30次MS/MS扫描，每次扫描覆盖100–1800 m/z范围、时长50 ms，总循环周期为1.8 s。选取电荷态为+2至+5、每秒计数不低于125的候选离子进行碎裂，在将其加入12 s的排除列表前，采集1张MS/MS谱图。实验采用滚动碰撞能量模式，碰撞能量扩散值设为15。质谱数据采集通过Analyst TF 1.7.1软件（AB Sciex）完成。
针对数据依赖采集（data dependent acquisition，DDA，SWATH采集），参数设置如下：先进行100 ms的飞行时间质谱（TOF MS）扫描，随后对m/z 400–1250质量范围设置200个可变SWATH窗口，每个窗口的累积时长为50 ms。MS/MS SWATH扫描窗口设置为5 Da、重叠1 Da，覆盖m/z 400–1250质量范围，且在质量范围两侧自动调整。总循环周期为9.6 s，采用滚动碰撞能量（CE）参数脚本自动控制碰撞能量。
采集得到的谱图通过嵌入于ProteinPilot 5.0.1软件（AB Sciex）中的Paragon算法（Paragon algorithm），比对至UniProt已审核数据库（UniProt reviewed database），搜索参数设置为：(i) 样品类型：鉴定；(ii) 半胱氨酸烷基化试剂：碘乙酰胺；(iii) 酶解方式：胰蛋白酶；(iv) 仪器型号：TripleTOF 6600；(v) 物种：智人（Homo sapiens）；(vi) 鉴定聚焦：生物学修饰；(vii) 蛋白检测阈值：>0.05（对应10%置信度）。为通过独立错误发现率（false discovery rate，FDR）分析实现曲线拟合，将蛋白检测阈值设置为最低水平以增加假阳性结果数量。该分析采用ProteinPilot软件自带的目标-诱饵库（target-decoy）方法完成，用于评估鉴定结果的质量。当鉴定到的蛋白及肽段的局部FDR达到1%时，视为阳性鉴定。
将得到的分组文件导入Peakview（v2.2.0，AB Sciex）中，以肽段置信度阈值99%、错误发现率<1%提取SWATH采集数据中的峰信息。随后将SWATH文件导出至MarkerView软件（版本1.3.0.1；AB Sciex），将单个肽段的峰面积归一化至所有检测到的肽段的峰面积总和。