Table 1.
Outcomes of different sample preparation strategies for measuring high mobility group 1 (HMGB1) protein in human plasma. Recovery was calculated by comparing the peak area response from the extracted ion chromatogram for the IKGEHPGLSIGDVAK peptide in processed samples along with the response in samples with pure recombinant HMGB1. The number of peptides and other proteins identified was found by searching the Global Proteome Machine (GPM) database [28,29]. Data files in the mascot generic format (MGF) can be downloaded from a data repository online [30].
| Sample Preparation Strategy | Recovery (%) | Results from Search in the GPM Database | File Name (.mgf) | |||
|---|---|---|---|---|---|---|
| Number of Specific HMGB1-Peptides | Total Proteins Identified | Rank# for HMGB1 | ||||
| A | Targeted extraction using anti-HMGB1 Ab | 7 | None | 5 | N/A | HMGB1_1 |
| Targeted extraction using anti-haptoglobin Ab | 16 | 2 | 5 a | 3 | HMGB1_2 | |
| B | Precipitation of HAPs using PCA | 35 | 6 | 61 | 28 | HMGB1_3 |
| C | Acid digestion of whole plasma | 12 b | 2 c | 68 c | 51 c | HMGB1_4 |
| Separation of proteins using HPLC | 33 | 14 | 19 | 3 | HMGB1_5 | |
| Pure recombinant HMGB1 | 100 | 17 | 1 | 1 | HMGB1_6 | |
a Contaminants not counted. b Recovery calculated using peak area response from extracted ion chromatogram for M(ox)SSYAFFVQTCR(NEM) peptide in processed samples compared to pure recombinant HMGB1. c Fractions between 19.8 min and 21.8 min. Ab, antibody; HAP, high-abundance proteins; HPLC, high performance liquid chromatography; PCA, perchloric acid.