Native lamin A/C brain proteome and novel partners in a mouse chronic inflammation model of human frailty

Tandem Mass Tag (TMT) proteomicsProtein extracts (40 ug total at 1 ug/ul in 1% SDS) were reduced with 15 uL of 15 mM DTT for 1 hour at 56°C, alkylated by adding 15 uL 100 mM iodoacetamide and incubating in the dark for 30 min, and then TCA/Acetone precipitated. The protein pellet from each sample was digested overnight at 37°C by adding 100 uL Trypsin/LysC mixture (40 ug proteases in 1.2 mL of 100 mM triethylammonium bicarbonate (TEAB; Promega #V5071) or approximately 3.33 ug protease per sample. Individual samples (40 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, the TMT reagents (0.8 ug vials) were allowed to come to room temperature before adding 41 uL anhydrous acetonitrile, then vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hour. The reaction was quenched by adding hydroxylamine (8 uL) to a final concentration of 5%. All TMT-labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid.Basic Reverse Phase (bRP) fractionation— Labeled peptide samples were fractionated by basic reverse phase (bRP) chromatography on Oasis HLB uElution plates (Waters). TMT labeled peptides (5%, approximately 20 ug) were bound to HLB resin in 10 mM triethylammonium bicarbonate (TEAB) buffer and step eluted with 0%, 5%, 10%, 25%, and 75% acetonitrile in 10 mM TEAB (0 and 5% fractions were combined). Fractions were dried by vacuum centrifugation.Mass Spectrometry Analysis— The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120Å, 75 um x 2 cm; YMC Co., LTD, Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um x 200 mm, 15 um, +/-1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120Å, www.dr-maisch.com) using 2-90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360-1700 m/z with a cycle time of 3 sec. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15s dynamic exclusion, with a scan range of 116m/z-2000m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 x 105 with 50 ms maximum injection time (IT) and 1 x 105 with 118ms maximum IT, respectively.Data analysis— Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted from raw MS data using spectrum selector with recalibration in Proteome Discoverer (PD) software (version 2.4.0.305, Thermo Scientific) and searched using Mascot (2.6.2; www.matrixscience.com) against a Mus musculus protein database (RefSeq2017_83, created 5/23/2019, containing 76,508 sequences. The following criteria were set for all database searches: (a) all species in database; (b) trypsin as the enzyme, (c) two missed cleavages allowed; (d) N-terminal TMT6plex and cysteine carbamidomethylation as fixed modifications; (e) lysine TMT6plex, methionine oxidation, serine, threonine and tyrosine phosphorylation, asparagine and glutamine deamidation, HexNAc on serine or threonine, as variable modifications; and (f) precursor and fragment ion tolerances were set to 5ppm and 0.03Da, respectively. Peptide identifications from Mascot searches were filtered at 5% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods. The protein intensities were reported as S/N of each peptide and relative protein comparisons were calculated using the peptide grouping in Proteome Discoverer. Quan value correction factors were used (Lot TK271715) with a co-isolation threshold of 30. Peptide abundances were normalized against a custom sequence .FASTA file containing only prelamin A (XP_006501136.1 PREDICTED: prelamin-A/C isoform X1 [Mus musculus]) to ensure there was no experimental bias in protein quantification that depended on the total amount of lamin A/C immunoprecipitated from each sample.

## 串联质谱标签（Tandem Mass Tag, TMT）蛋白质组学实验流程
### 样品前处理与酶解
总蛋白提取液（总量40 μg，浓度1 μg/μL，溶于1%十二烷基硫酸钠（SDS）中）经以下步骤处理：加入15 μL 15 mM二硫苏糖醇（DTT），于56℃还原1小时；随后加入15 μL 100 mM碘乙酰胺进行烷基化，避光孵育30分钟；再经三氯乙酸/丙酮（TCA/Acetone）沉淀。将每份样品的蛋白沉淀用100 μL胰蛋白酶/赖氨酸C（Trypsin/LysC）混合液进行酶解，于37℃孵育过夜；该混合液配置为：40 μg蛋白酶溶于1.2 mL 100 mM三乙胺碳酸氢盐（TEAB；Promega #V5071），即每份样品约对应3.33 μg蛋白酶。

### 肽段标记
每份40 μg的肽样品按照厂商说明书，使用专属的同量异位质量标签试剂（TMT 10重标记试剂盒，Thermo Scientific）进行标记；TMT试剂与肽样品的配对及标记顺序均经过随机化处理。简要操作如下：将0.8 μg/瓶的TMT试剂恢复至室温后，加入41 μL无水乙腈，涡旋混匀并离心；将整瓶TMT试剂加入至100 μg肽样品中，于室温反应1小时；随后加入8 μL羟胺使终浓度达5%以终止反应。将所有标记完成的TMT样品混合后，经真空离心干燥去除全部溶剂。

### 碱性反相分级
标记后的肽样品通过Oasis HLB洗脱微孔板（Waters公司）进行碱性反相（bRP）色谱分级。取5%的TMT标记肽（约20 μg），于10 mM三乙胺碳酸氢盐（TEAB）缓冲液中结合至HLB树脂，随后使用含0%、5%、10%、25%、75%乙腈的10 mM TEAB缓冲液进行分步洗脱（0%与5%洗脱组分合并收集）；各级分经真空离心干燥。

### 质谱分析
将肽分级组分重悬于20 μL含2%乙腈的0.1%甲酸溶液中；取约0.5 μg（2 μL）上样至C18捕集柱（S-10 μm，120Å孔径，75 μm×2 cm；YMC Co., LTD, 日本京都），随后通过自制填充的PicoFrit色谱柱（75 μm×200 mm，15 μm粒径，尖端孔径±1 μm，New Objective公司）进行分离，色谱填料为ReproSil-Pur C18-AQ（3 μm粒径，120Å孔径，www.dr-maisch.com），使用EasyLC nanoLC 1000液相色谱系统（Thermo Scientific），以300 nL/min流速通过2%-90%乙腈梯度洗脱120分钟。洗脱的肽段经2.0 kV电压直接喷雾进入Orbitrap Fusion Lumos质谱仪（Thermo Scientific）。

质谱采集参数如下：全扫描（MS1）采集范围为360-1700 m/z，循环周期为3秒。以0.7 Da窗口分离前体离子，采用高能碰撞解离（HCD）模式，碰撞能量为39%，动态排除时长15秒，碎片离子扫描范围为116-2000 m/z。前体离子与碎片离子的分析分辨率分别设为120,000与30,000；自动增益控制（AGC）目标值分别为4×10^5和1×10^5，最大注入时间（IT）分别为50 ms和118 ms。

### 数据分析
使用Proteome Discoverer（PD）软件（版本2.4.0.305，Thermo Scientific）中的Spectrum Selector工具，对原始质谱数据进行提取并完成重校准，得到前体（MS1）与碎裂（MS2）谱图中的同位素解析质量峰；随后通过Mascot搜索引擎（版本2.6.2；www.matrixscience.com），以小鼠（Mus musculus）蛋白质数据库（RefSeq2017_83，创建于2019年5月23日，包含76,508条序列）进行搜库。

所有搜库均设置如下参数：(a) 数据库内所有物种；(b) 酶解酶为胰蛋白酶；(c) 允许最多2个漏切位点；(d) N端TMT6plex标记与半胱氨酸氨基甲酰化为固定修饰；(e) 赖氨酸TMT6plex标记、甲硫氨酸氧化、丝氨酸/苏氨酸/酪氨酸磷酸化、天冬酰胺/谷氨酰胺脱酰胺、丝氨酸/苏氨酸HexNAc糖基化为可变修饰；(f) 前体离子与碎片离子的质量偏差容忍度分别设为5 ppm与0.03 Da。

通过串联反向数据库搜库，使用Proteome Discoverer将Mascot鉴定得到的肽段以5%错误发现率（FDR）作为置信阈值进行过滤。Proteome Discoverer仅保留不同提取方法中，与同一肽段谱图匹配的Mascot得分最高的肽段鉴定结果。蛋白质强度以每条肽段的信号噪音比（S/N）表示，相对蛋白质定量通过Proteome Discoverer中的肽段分组功能进行计算。使用批次为TK271715的定量值校正因子，共分离阈值设为30。肽段丰度以仅包含前层粘连蛋白A（XP_006501136.1 预测：前层粘连蛋白-A/C亚型X1 [Mus musculus]）的自定义FASTA序列文件进行归一化，以确保蛋白质定量不存在因每份样品中免疫沉淀的层粘连蛋白A/C总量不同而产生的实验偏差。