| Metadata |
| datasetIdentifier | PASS01453 |
| datasetType | SRM |
| submitter | Yong Zhou <yzhou@systemsbiology.org> |
| submitter_organization | Institute for Systems Biology |
| lab_head_full_name | Leroy Hood |
| lab_head_email | lhood@systemsbiology.org |
| lab_head_organization | Institute for Systems Biology |
| lab_head_country | United States |
| datasetTag | Acute_Lyme_SRM_ISB |
| datasetTitle | Measurement of potentially diagnostic organ-specific and acute-phase blood protein level changes in early Lyme disease |
| publicReleaseDate | 2019-09-30 00:00:00 |
| finalizedDate | 2019-10-11 10:56:35 |
| summary | To identify blood proteins with altered abundances in patients with early stage Lyme disease as compared to healthy controls with the intent of identifying potential Lyme diagnostic markers, selected proteins from two categories were monitored by SRM: 1) key innate immunity regulators including acute phase proteins, reflecting the fact that LD is an acute inflammatory disease; and 2) organ-specific blood proteins predominantly expressed in organs and tissues known to be affected by B. burgdorferi, i.e., liver, heart, brain and skin. In all, 174 peptides (representing 124 human proteins) were measured in individual serum samples from 2 independent Lyme disease patient cohorts. Each sample set contains 30-40 confirmed Lyme disease patients (each with 4 time points) and 20 healthy controls (each with 2 time points) from baseline to 4-6 years years post-treatment. 90 proteotypic peptides from 73 targeted proteins were successfully detected and quantified. Six proteins (APOA4, C9, CRP, CST6, PGLYRP2 and S100A9) were confirmed to show significantly altered serum levels in patients at time of presentation. A 9-protein panel was also identified and verified. |
| contributors | Yong Zhou, Shizhen Qin |
| publication | Zhou, Y, et al. Measurement of organ-specific and acute-phase blood protein levels in early Lyme disease, Journal of Proteome Research. doi: 10.1101/795344 |
| growth | n/a |
| treatment | n/a |
| extraction | n/a |
| separation | An Mars14 LC depletion column was applied to remove the 14 top abundant proteins from serum. |
| digestion | Depleted sera were collected for trypsin digestion:
The flow-through fractions (1.2 mL) were collected and concentrated to 200 µL using Amicon Ultra Centrifugal Filters (4 mL 3K MWCO, MilliporeSigma, Burlington, MA). BCA protein assay was performed to measure protein concentrations before and after depletion. Proteins were denatured in 50% (v/v) TFE (2,2,2-Trifluoroethanol) at 37℃ for 30 min before reduction (TCEP), alkylation (iodoacetamide) and digestion with trypsin (1:25 trypsin/protein ratio) for 14 hours. After desalting using 1 cc C18 and MCX cartridges (Waters, Milford, MA), the proper amounts of stable C13N15 isotope-labeled (SIL) synthetic standards for target proteins were spiked into experimental samples before analysis in Agilent 6490 triple-quadrupole mass spectrometers (Agilent, Santa Clara, CA). A yeast protein (enolase 1, P00924) was spiked in as internal standard before depletion. |
| acquisition | Serum samples were analyzed in a triple-quadrupole mass spectrometer (Agilent 6490, Agilent, Santa Clara, CA) integrated with a nanospray ion source and Chip Cube nano-HPLC. Three to four pairs of light (endogenous) and heavy (stable C13N15 isotope-labeled (SIL) synthetic standards) transitions were monitored for each target peptide. A 90-min gradient of acetonitrile from 3% to 40% was used to elute peptides from a high-capacity nano-HPLC Chip (160 nL, 150 mm X 75 μm ID, Agilent, Santa Clara, CA) as described elsewhere 26. Other settings included operating the nano-HPLC separation chip at 0.3 L/min nano pump flow rate, 2 g peptide loading amount, 1820 V capillary voltage and Dynamic MRM with 200 Delta EMV (+). Duplicate runs were performed for each sample. |
| informatics | Raw SRM mass spectrometry data were processed using the Skyline targeted proteomics environment. For each peptide, the total peak area and L/H ratio of all monitored transitions were calculated and manually checked before export. The quantifier transition(s) were selected and used in further calculations in cases of high background and overlapping peaks from co-eluted peptides. Although the same amount of total peptide (2 μg) from each depleted sample was loaded into the mass spectrometer, the L/H ratios were adjusted by serum volume such that the relative peptide abundances from the same volume of serum were compared. The peptide L/H ratios were then log2-transformed for a more normal-like distribution. The peptide levels were normalized against the mean of the 20 control samples from two time points since there was no significant difference in controls between the two time points. Significantly differentially expressed proteins were identified by both Student’s t-test and multivariate analysis. |
| instruments | Agilent 6490 |
| species | Human |
| massModifications | static: C+57.021464,
variable: K+8.014199, R+10.008269 |
