PRIDE Assigned Tags:Biomedical Dataset
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Urinary polypeptide ETD/CID analysis, part 3
Urine as a biofluid is commonly used in clinical diagnostics, including those performed during pregnancy. Urine is a rich source of polypeptides and polypeptidic protein degradation products, which have been filtered from blood plasma, thus urine has potential as a source for novel clinical diagnostics in disease. In this study, we examine the urinary peptidome from normal healthy women during pregnancy, and demonstrate ready observation of large polypeptide. We utilise the dissociation method, electron transfer dissociation (ETD) to increase the identification rate of the peptides present within these samples, as the polypeptide species observed in these samples are large and highly charged. An increase in the number of peptides whose identities could be ascribed using routine database searching methods was enabled via the use of ETD.
Sample Processing Protocol
Urine samples were thawed and spun to pellet urinary tract cellular debris (3,000 rpm, 4°C, 20 min). Peptides were separated from proteins (arbitrary NMWCO of 10kDa) using centrifugal concentrators (Vivaspin 20, Sartorius AG), with the spin through peptide fraction being subjected to solid phase extraction (SPE) to concentrate and clean up the peptides. SPE was performed using HLB cartridges (Aldrich), which were used according to manufacturers’ instructions, eluting bound materials using 60% acetonitrile, 0.1% trifluoracetic acid. SPE-eluted components were subjected to strong cation exchange treatment to segregate bile components from peptides. Peptides were eluted in 500 mM ammonium acetate in 20 % ACN, volatile buffer components were then removed by vacuum centrifugation prior to peptides being subjected to LC-MS/MS analysis. LC-MS/MS was performed on an orbitrap instrument (LTQ Orbitrap XL MS, ThermoFisher Scientific, San Jose, CA) equipped with ETD source, coupled to a nanoAcquity HPLC (Waters Corp, Milford, MA); separation was performed over 100min gradients from 0-80% acetonitrile on a BEH column (75um x 200mm, 1.7um particle size). A ‘Top 3’ data dependent acquisition, whereby the three most abundant multiply-charged precursors from a given survey spectrum, collected in the orbitrap mass analyser (resolution 30,000), were selected for independent fragmentation by both CID and ETD within the linear ion trap (AGC 30,000). CID was performed using an isolation width of 2amu, normalised collision energy of 35, activation Q 0.25 and activation time of 30 msec. ETD was performed with an isolation width of 3amu and activation time of 200 msec and anion AGC of 100,000; supplemental activation was applied. Dynamic exclusion parameters were: repeat count 1, duration 30 sec, exclusion duration 60 sec, exclusion mass width -0.1- +1.1 amu, exclusion list 500 entries.
Data Processing Protocol
Raw mass spectra were processed using a custom script written at UCSF, PAVA 29 to generate mgf-formatted text files, and subjected to Mascot searching (Matrix Science, London). Search parameters were: SwissProt database (download date Feb 19 2014) with taxonomy restricted to homo sapiens (20,271 sequences), no enzyme filter applied, precursor ion tolerance 5ppm, product ion tolerance 0.6Da, search type CID+ETD, decoy search applied. Methionine oxidation was allowed as a variable modification. Mascot identification data were imported to Scaffold Q+ V 4.3.3 (Proteome Software, Portland, OR), applying peptide and protein thresholds of 95% confidence, with a minimum of 1 peptide for identification.