Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.
Posttranslational modification of proteins by phosphorylation plays a complex and critical role in the regulation of numerous biological processes. In recent years, large efforts have been devoted to global-scale analysis of protein phosphorylation sites using various phosphoproteomics methodologies [
Recent studies of phosphoproteomics of biological fluids include serum and plasma [
Human bronchoalveolar lavage fluid (BAL) is a proximal fluid commonly used for diagnosis of lung diseases including chronic obstructive pulmonary disease (COPD) and lung cancer. Procurement of clinical BAL specimens involves washing of the epithelial lining of the lung with saline using a fiberoptic bronchoscope. Molecular composition of BAL reflects the status of the respiratory tract, and analysis of human BAL composition at the molecular level therefore provides an attractive way towards improved understanding of disease mechanisms or discovery of biomarker signatures that are directly relevant to specific lung diseases. The proteome of human BAL has been studied numerous times in the context of various lung diseases [
In this study, we undertook a pilot interrogation of the human BAL phosphoproteome. Our ongoing research program focuses on proteomics of human BAL [
The human BAL specimens were provided by Chiesi Farmaceutici, Parma Italy; the project was approved by the IRB at The University of Tennessee Health Science Center. The human BAL samples were obtained from subjects without clinical diagnosis of COPD or lung cancer. Information on the characteristics of the BAL specimen donors is listed in Table
Characteristics of BAL specimen donors.
Donor | Disease status | Gender | Age |
---|---|---|---|
1 | control | F | 48 |
2 | control | F | 68 |
3 | control | F | 58 |
4 | control | F | 75 |
5 | control | F | 58 |
6 | control | F | 64 |
7 | control | F | 63 |
8 | control | F | 60 |
9 | control | F | 65 |
10 | control | F | 73 |
Prior to analysis, the BAL samples were centrifuged to remove cell debris. Processing of each sample included removal of salts and depletion of overabundant contaminant proteins. Desalting was performed by ultrafiltration with spin concentrators (MW cutoff of 5,000 Da). The samples in the concentrators were centrifuged (25 min; 5,000 g; 4°C) to produce
Albumin and five other high-abundance proteins were removed with the Hu-6 Multiple Affinity Removal System (MARS) spin cartridge (Agilent) following procedure provided by the manufacturer. After MARS depletion, the samples were desalted by ultrafiltration as described above. Protein concentration before and after MARS depletion was determined with the micro BCA assay (Pierce). After pooling, the final protein content was 450
The proteins were digested with trypsin using an in-solution digestion procedure. Briefly, the dried proteins in each pooled sample were redissolved in 45
After digestion, the mixture was acidified with TFA and subjected to solid phase extraction using a home-made SPE minicolumn packed with C18 stationary phase. After elution from the minicolumn, the sample was dried and the redissolved in 90% water/10% acetic acid, as required for immobilized metal ion affinity chromatography (IMAC).
The IMAC procedure, which serves to enrich the proteolytic digests for phosphopeptides, was performed with the Phosphopeptide Isolation Kit (gallium/IDA, Pierce). Each BAL peptide digest was applied to the column, and the phosphopeptides were bound by incubation at room temperature for 1 h. The column was washed with the following solutions: 40
The LC-MS/MS analyses were performed with an LTQ linear ion trap mass spectrometer (Thermo Electron) that was interfaced with a nano-LC system (Dionex). The IMAC-enriched peptide digests were loaded onto a fused-silica microcapillary column/spray needle (Picofrit, 15 cm length, 75
The LC-MS/MS datasets were used to search the UniProt database (subset of human proteins) using TurboSEQUEST search engine that was part of Bioworks 3.2 (Thermo Electron). The following parameters were used in the searches: full-trypsin specificity, dynamic modifications of phosphorylated S, T, and Y (+80.0), and dynamic modifications of oxidized M (+16.0). The search results were filtered to include peptides retrieved XCorr values ≥2.00, and 3.50 for doubly and triply charged precursor ions, respectively. All MS/MS spectra for the individual phosphopeptides that passed this initial filtering were inspected manually. This manual validation checked for the presence of a product ion that corresponds to the neutral-loss of phosphoric acid ([M+2H-98]2+ for doubly charged ions or [M+3H-98]3+ for triply charged ions); and for coverage of the phosphopeptide sequence by the b- and/or y product-ion series. Assignments of the sites of phosphorylation were verified by inspecting the b- and/or y-product ions that flanked the phosphorylation site assigned by the search engine. Data from analyses of Pool 1 and Pool 2 were combined to produce the final phosphoprotein panel. Additional information about the phosphorylation sites/phosphoproteins was obtained from the UniProt annotations, the Phosphosite knowledgebase (
For this pilot study, a simple gel-free bioanalytical strategy was employed. The general outline of the bioanalytical workflow is shown in Figure
Flowchart depicting the bioanalytical workflow used for BAL phosphoproteome mapping. Abbreviations: Multiple Affinity Removal System—MARS; immobilized metal ion affinity chromatography—IMAC.
Representative MS/MS spectrum obtained in analyses of IMAC-enriched digests of depleted BAL proteomes. The spectrum displays a prominent product ion at
Each of the two pooled BAL samples that were analyzed produced a set of 13 phosphoproteins. Five of these phosphoproteins were common to both samples; in addition, each sample yielded a unique group of phosphoproteins. This is not unexpected given the large biological variability associated with clinical specimens, and variable phosphoprotein signatures have been also observed for other clinical samples [
Phosphopeptides and phosphoproteins characterized in human BAL.
Database accession code | Entry name | Protein name |
Siteb |
---|---|---|---|
(1) P08670 | VIME_HUMAN | Vimentin | |
QVQS*LTCEVDALK | S325 | ||
(2) P02794 | FRIH_HUMAN | Ferritin heavy chain | |
KM#GAPESGLAEYLFDKHTLGDS*DNES | S179 | ||
KMGAPESGLAEYLFDKHTLGDS*DNES | S179 | ||
MGAPESGLAEYLFDKHTLGDS*DNES | S179 | ||
HTLGDS*DNES | S179 | ||
KMGAPESGLAEYLFDKHTLGDSDNES* | (S183) | ||
MGAPESGLAEYLFDKHTLGDSDNES* | S183 | ||
(3) P30086 | PEBP1_HUMAN | Phosphatidylethanolamine-binding protein 1 | |
NRPTS*ISWDGLDSGK | S52 | ||
(4) P13796 | PLSL_HUMAN | Plastin-2 | |
GS*VSDEEM#M#ELR | S5 | ||
GS*VSDEEMM#ELR | S5 | ||
GS*VSDEEMMELR | S5 | ||
EGES*LEDLMK | S257 | ||
(5) Q9H3Z4 | DNJC5_HUMAN | DnaJ homolog subfamily C member 5 | |
S*LSTSGESLYHVLGLDK | (S8) | ||
(6) P27816 | MAP4_HUMAN | Microtubule-associated protein 4 | |
DVT*PPPETEVVLIK | T521 | ||
(7) P21333 | FLNA_HUMAN | Filamin-A | |
RAPS*VANVGSHCDLSLK | S2152 | ||
CSGPGLS*PGMVR | S1459 | ||
(8) P08575 | PTPRC_HUMAN | Receptor-type tyrosine-protein phosphatase C | |
NRNS*NVIPYDYNR | S973 | ||
(9) P01042 | KNG1_HUMAN | Kininogen-1 | |
ETTCSKES*NEELTESCETK | S332 | ||
(10) P02765 | FETUA_HUMAN | Alpha-2-HS-glycoprotein | |
CDSSPDS*AEDVRK | S138 | ||
CDSSPDS*AEDVR | S138 | ||
(11) Q15637 | SF01_HUMAN | Splicing factor 1 | |
TGDLGIPPNPEDRS*PS*PEPIYNSEGK | S80; S82 | ||
(12) Q9UK76 | HN1_HUMAN | Hematological and neurological expressed 1 protein | |
RNS*SEASSGDFLDLK | (S87) | ||
(13) Q7Z3D4 | LYSM3_HUMAN | LysM and putative peptidoglycan-binding domain-containing protein 3 | |
S*TSRDRLDDIIVLTK | (S53) | ||
(14) P02671 | FIBA_HUMAN | Fibrinogen alpha chain | |
PGSTGTWNPGS*SER | S364 | ||
(15) P09651 | ROA1_HUMAN | Heterogeneous nuclear ribonucleoprotein A1 | |
SES*PKEPEQLR | (S6) | ||
(16) P51858 | HDGF_HUMAN | Hepatoma-derived growth factor | |
AGDLLEDS*PKRPK | S165 | ||
RAGDLLEDS*PK | S165 | ||
AGDLLEDS*PK | S165 | ||
GNAEGSS*DEEGKLVIDEPAK | (S133) | ||
(17) Q9H2C0 | GAN_HUMAN | Gigaxonin | |
FGAVACGVAMELY*VFGGVR | Y471 | ||
(18) Q13637 | RAB32_HUMAN | Ras-related protein Rab-32 | |
DSS*QSPSQVDQFCK | (S152) | ||
(19) P35579 | MYH9_HUMAN | Myosin-9 | |
KGAGDGS*DEEVDGK | S1943 | ||
(20) P07900 | HS90A_HUMAN | Heat shock protein HSP 90-alpha | |
DKEVS*DDEAEEK | S231 | ||
(21) P08238 | HS90B_HUMAN | Heat shock protein HSP 90-beta | |
IEDVGS*DEEDDSGKDKK | S255 | ||
IEDVGS*DEEDDSGKDK | S255 | ||
IEDVGS*DEEDDSGK | S255 |
aSTY* denotes phosphorylated amino acid. M# denotes oxidized methionine.
bPhosphorylation sites were assigned based on MS/MS product ions. Parentheses indicate cases where an alternative site is possible.
Since the focus of our pilot study reported here was on first description of the human BAL phosphoproteome, the scope of the study was limited to qualitative analyses of a small number of specimens from female donors only. This initial examination was not intended to characterize phosphoprotein biomarkers associated with a specific lung disease but to initiate the building of a detailed phosphoprotein/phosphosites catalog as a starting point for future differential phosphoproteomics efforts. In terms of the size of our initial BAL phosphoprotein panel, our results are comparable, for example, to a CSF phosphoproteome study that revealed 44 phosphoproteins [
Bronchoalveolar lavage samples components of the epithelial lining fluid, and proteins that are found in BAL are of diverse origin [
Tissue expression and subcellular location for proteins from our panel. This information was compiled from Human Protein Atlas (HPA) and from Ingenuity Pathway Analysis.
No. | Database code | Entry name | Protein name | Tissue expression in the lung |
Subcellular location | ||
---|---|---|---|---|---|---|---|
Pneumocytes | Macrophages | Other tissues | |||||
1 | P08670 | VIME_HUMAN | Vimentin | Y |
Y |
Y | Cytoplasm |
2 | P02794 | FRIH_HUMAN | Ferritin heavy chain | N | Y |
Y | Cytoplasm |
3 | P30086 | PEBP1_HUMAN | Phosphatidylethanolamine-binding protein 1 | Y |
Y |
Y | Cytoplasm |
4 | P13796 | PLSL_HUMAN | Plastin-2 | N | Y |
Y |
Cytoplasm |
5 | Q9H3Z4 | DNJC5_HUMAN | DnaJ homolog subfamily C member 5 | Y |
Y |
Y | Plasma membrane |
6 | P27816 | MAP4_HUMAN | Microtubule-associated protein 4 (MAP 4) | Y |
Y |
Y | Cytoplasm |
7 | P21333 | FLNA_HUMAN | Filamin-A | Y |
Y |
Y | Cytoplasm |
8 | P08575 | PTPRC_HUMAN | Receptor-type tyrosine-protein phosphatase C | N | Y |
Y |
Plasma membrane |
9 | P01042 | KNG1_HUMAN | Kininogen-1 | N | N | Y |
Extracellular space |
10 | P02765 | FETUA_HUMAN | Alpha-2-HS-glycoprotein | N | Y |
Y |
Extracellular space |
11 | Q15637 | SF01_HUMAN | Splicing factor 1 | Y |
Y |
Y | Nucleus |
12 | Q9UK76 | HN1_HUMAN | Hematological and neurological expressed 1 protein | Nucleus |
|||
13 | Q7Z3D4 | LYSM3_HUMAN | LysM and putative peptidoglycan-binding domain-containing protein 3 | Y |
Y |
Y | unknown |
14 | P02671 | FIBA_HUMAN | Fibrinogen alpha chain | N | Y |
Y |
Extracellular space |
15 | P09651 | ROA1_HUMAN | Heterogeneous nuclear ribonucleoprotein A1 | Y |
Y |
Y | Nucleus |
16 | P51858 | HDGF_HUMAN | Hepatoma-derived growth factor; | Y |
Y |
Y | Extracellular space |
17 | Q9H2C0 | GAN_HUMAN | Gigaxonin | N | Y |
Y | Cytoplasm |
18 | Q13637 | RAB32_HUMAN | Ras-related protein Rab-32 | Y |
Y |
Y | Cytoplasm |
19 | P35579 | MYH9_HUMAN | Myosin-9 | Y |
Y |
Y | Cytoplasm |
20 | P07900 | HS90A_HUMAN | Heat shock protein HSP 90-alpha | N | N | Y |
Cytoplasm |
21 | P08238 | HS90B_HUMAN | Heat shock protein HSP 90-beta | Y |
Y |
Y | Cytoplasm |
Subcellular location distribution of the characterized proteins; compiled from Ingenuity.
The BAL phosphoprotein panel (Table
For example, ferritin is an important mediator of iron homeostasis, and increased levels of ferritin have been found in the lavage of smokers [
Another phosphoprotein found in the present study is the actin-bundling protein plastin-2. Phosphorylation of plastin-2 modulates its function in the assembly of actin networks, and it is associated with leukocyte activation in response to various stimuli [
In conclusion, this study presents novel findings towards description of the human BAL phosphoproteome. Since aberrant protein phosphorylation associated with specific lung diseases could potentially be reflected as alterations in BAL phosphoproteins, this study lays an important foundation for future differential phosphoprotein profiling for biomarker discovery.
This study has been funded by Chiesi Farmaceutici. Funds for the LTQ mass spectrometer have been provided in part by the NIH Shared Instrumentation Grant S10RR16679 (to D.M.D).