A questionnaire was sent to healthy plasma donors in the Bama longevity area, and the number of long-lived immediate family members (>= 85 years old) within three generations in the donors were investigated. A donor with at least three long-lived immediate family members was defined as the offspring of longevous family, and donors without an immediate family member >= 85 years old within three generations were offsprings of non-longevity families.
In total, 33 offsprings of longevous families were recruited as case studies (Longevous group). And also 33 ABO-, age- and gender-matched offsprings of non-longevity families were recruited as controls (Normal group). The inclusion criteria of the participators were recruited as previously described and apheresis plasma of the participators was used in this study. Briefly, all participators were over 18 years of age, healthy and unrelated. People who had prior history of thrombus or hemorrhage, usage of oral anticoagulation therapy, hepatic disease, HIV infection, pregnancy, diabetes, cardiopathy, hypertension, renal insufficiency, respiratory diseases and others were excluded from this study. Pooled plasma samples were generated by combining equal volumes of each 11 individual plasma samples from each group. The basic information of the volunteers was shown in Table 1. This study was approved by the Ethics Committee of the Institute of Blood Transfusion (No. 201716). In accordance with the Declaration of Helsinki, all participants gave informed consent prior to their entering the study. 
The high abundance proteins that account for approximately 80% of the total plasma protein concentration were removed from the pooled plasma samples, using a ProteoMiner  Protein Enrichment Introductory Large-Capacity Kit (Bio-Rad, Richmond, USA) according to the manufacturer's instructions. After being reduced with 5 mM DL-dithiothreitol for 30 min at 56  C and alkylated with 11 mM iodoacetamide for 15 min in darkness at room temperature, the protein sample were digested with trypsin at a trypsin/protein mass ratio of 1:50 overnight in the first stage and 1:100 for 4 h in the second stage. Approximately 100 microg protein for each sample was digested with trypsin for the following experiments. Tryptic peptides were desalted by Strata X C18 SPE column (Phenomenex, CA, USA) and labeled with a 6-plex TMT kit (Thermo Fisher Scientific, CA, USA) according to manufacturer's instructions. The 6 labeled samples then fractionated by high pH reverse-phase HPLC using 300Extend C18 column (5 microm particle size, 4.6 mm ID, 250 mm length, Agilent, CA, USA). Peptides were eluted with a gradient of acetonitrile (8% to 32%) in 10 mM ammonium bicarbonate (pH 9.0) over 60 min. At last, 60 fractions were collected, and they were combined into 18 fractions and dried by vacuum centrifuging.
Samples were measured using LC-MS instrumentation consisting of an EASYnLC 1000 ultra-high-pressure system coupled via a nano-electrospray ion source to a Q Exactive  Plus (Thermo Fisher Scientific). Purified peptides were separated on a reversed-phase analytical column (150 mm length, 75 mum ID). For each LC-MS/MS analysis, the electrospray voltage applied was 2.0 kV. Intact peptides were detected in the orbitrap at a resolution of 70,000, and ion fragments were detected in the orbitrap at a resolution of 17,500. In the MS survey scan, a data-dependent mode with an automatic alteration (1 MS scan followed by 20 MS/MS scans) was used for the top 20 precursor ions above a threshold ion count of 5 x 104 with 30 s dynamic exclusion. Automatic gain control was used to prevent overfilling of the orbitrap; 5 x 104 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800, and the fixed first mass was set as 100 m/z.
MS/MS data was analyzed by MaxQuant software with integrated Andromeda search engine (Version 1.5.2.8). The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and that for fragment ions was set as 0.02 Da. False discovery rate (FDR) was adjusted to < 1% and minimum score for peptides was set > 40. Only unique peptides were used for protein quantification. And for protein quantification method, TMT 6-plex was selected in Mascot. FDR was adjusted to < 1% at protein, peptide and peptide spectrum match (PSM) level. Gene Ontology (GO) annotation (the biological process, cellular component and molecular function) of the differentially expressed proteins were derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). PSORT/PSORT II and SubLoc (http://www.bioinfo.tsinghua.edu.cn/SubLoc/) were used to predict subcellular localization of all identified DEPs. A p value < 0.05 was used as the threshold to determine the significant enrichments of GO annotation.
The HuProt  V3.1 microarray (CDI Laboratories, Inc. Mayaguez, USA) composed of about 17,000 recombinant human proteins was used in this study. All of the recombinant human proteins were generated by the Saccharomyces cerevisiae expression system and carried an N-terminal glutathione S-transferase (GST) tag. Each protein was spotted in duplicate. The microarray was firstly blocked with blocking buffer (5% BSA in 0.1 mol/l TBS-T, pH = 7.5) at room temperature for 1.5 h. Then it was washed with with TBS-T and then Milli-Q water for 10 min respectively. The six pooled plasma samples of two groups were diluted 1: 2000 in 0.01 mol/l Tris-Buffered saline containing 0.1% (v/v) Tween 20 detergent (TBS-T, pH = 7.5) with 5% BSA. Five ml of diluted plasma were added and incubated with microarray under a glass coverslip at room temperature for 1 h. After rinsing with TBS-T and then Milli-Q water, Cy3-conjugated anti-human IgG diluted 1:2000 in TBS-T was added to the washed microarray and incubated in darkness at room temperature for 1 h. The microarray was then washed again as described above. After drying by centrifugation (ChipMate PMC-082, Tomy, Japan), microarray was subjected for scanning with the Axon GenePix 4000B microarray scanner (Molecular Devices, CA, USA) using an excitation wavelength of 523 nm and an emission filter specifically for cy3 fluorescent dye. The optical signal was detected by a photomultipliers tube (PMT).
Human proteome microarray data analysis GenePix Pro 6.0 software (Molecular Devices, CA, USA) was used to obtain microarray signal intensity. The foreground median intensity (F median) and background median intensity (B median) for each spot on the microarray images were acquired. To quantify the signal intensity for each spot, we calculated the signal intensity for each spot, which was defined as the foreground median intensity divided by its local background median intensity (F median/B median). The intra-array signal intensity normalization and positive hits identification were taken as described in the previous reports. Briefly, the signal intensities of the spots within a protein microarray were normalized by setting the median intensity of that microarray equal to 1. To identify autoantibodies that bind to a protein on microarray (positive hits), an intensity cutoff value needed to be assigned for each microarray. We used I to denote the normalized intensity of a spot (control spots omitted) on the protein microarray. And Meani denote the average of I (control spots omitted) on a protein microarray. The standard deviation (SD) away from the mean of the signal intensities for all the spots in a microarray was calculated. A cutoff was defined as Meani + 6SD, and spots producing a signal intensity greater than the cutoff were identified as "positive hits." Moreover, each protein was printed in duplicate on a microarray, proteins and controls were considered as a positive feature only when both of their duplicate spots were simultaneously judged as a positive hit. T-test was chosen to assess the differential significance for each protein between the two groups based on signal value. The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 were used for the functional category analyzation of the identified autoantigens, subcellular localization prediction and GO annotation of the different autoantigens between groups.
The datasets supporting the conclusions of this article are included within the article and its additional files.
This study was approved by the Ethics Committee of the Institute of Blood Transfusion (No.201716). In accordance with the Declaration of Helsinki, all participants gave informed consent prior to their entering the study.
All authors read and approved the final manuscript.