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You are ‘free’ to choose most instrument method parameters; nevertheless, there are some rules that participants should follow in order to make posterior data analysis more feasible.
	Rule 1: Liquid Chromatography
	Rule 2: MS acquisition method
	Rule 3: The data file
	Rule 4: Occurrence report
Rule 1: Liquid Chromatography We have devised general a LC gradient for sample and blank runs. Although we understand that all laboratories may not be able to strictly follow these directions, we ask you to attempt to reproduce it as close as possible. The most important point to keep in mind is the elution gradient itself – from 2 to 30% B in 110 minutes. Note 1. Column equilibration is performed at the end of the run. If you prefer to equilibrate at the beginning of the run, feel free to adapt it. Note 2. Some laboratories may also prefer to use variable flow rate (e.g., increasing flow rate when it is at 90%B). Note 3. If you prefer to inject in full loop, make appropriate sample dilution so as the final amount injected is 200 ng. Note 4. Most LC-MS systems present some delay from the start of the gradient to the time that the peptides are injected into the MS system. To avoid collection of unnecessary data, laboratories often set a “Start delay” and end the acquisition before the end of the LC run. For example, in our system, we do not acquire the first 15 minutes and end acquisition after 140 minutes. 150-min HPLC gradient for yeast samples Time (min) Flow (nl/min) %B Initial 250 2 1 250 2 110 250 30 120 250 40 125 250 90 130 250 90 132 250 2 150 250 2 Mobile phase A: 0.1% (v/v) formic acid in water Mobile phase B: 0.1% (v/v) formic acid in acetonitrile 60 min HPLC for blank runs Time (min) Flow (nl/min) %B Initial 250 2 1 250 2 25 250 30 30 250 60 35 250 90 45 250 90 47 250 2 60 250 2 Mobile phase A: 0.1% (v/v) formic acid in water Mobile phase B: 0.1% (v/v) formic acid in acetonitrile Run order for study A and B. Samples should be run on the following order: Run number Sample HPLC run 1 Yeast peptide digest sample (study A) 150-min 2 Blank 1 (study A) 60-min 3 Blank 2 (study A) 60-min 4 Yeast peptide digest sample (study B) 150-min 5 Blank 1 (study B) 60-min 6 Blank 2 (study B) 60-min Run order for the study A. Samples should be run on the following order: Run number Sample HPLC run 1 Yeast peptide digest sample (study A) 150-min 2 Blank 1 (study A) 60-min 3 Blank 2 (study A) 60-min
Rule 2: MS acquisition method You are free to choose the MS acquisition parameters. The only exception to this rule is the ‘data type for MS scan’; that is, MS1 scans must be acquired in profile mode. The remaining parameters such as number of scan events, AGC, exclusion time and so on are up to you. The most important thing to note here is that the MS acquisition method MUST NOT be modified throughout the duration of this study. This is extremely important since results will be compared over time, and any modifications to the acquisition method will introduce unwanted bias not related to laboratory reproducibility/repeatability. After the first sample analysis, all participants must fill out a SOP checklist (excel file here: http://www.proteored.org/PMEQC/ProteoRed SOP Checklist.xlsx) which summarizes most relevant LC-MS system parameters. Because the methods should not change throughout the study, you only need to fill out this checklist once.
Rule 3: The data file Every week (or every two weeks), you should upload your .raw file in our ftp (ftp://estrellapolar.cnb.csic.es) The rule on uploading the data is to use your three-letter code followed by the study type A or B (with its digestion number, from 1 to 6), and the date of analysis (YYYY-MM-DD). For example, if you choose PCB as your three-letter code, you will name your files as follows: 1st week PCB_A_20110509.raw (study A sample) PCB_blank1A_20110509.raw (first blank in the run order after study A sample) PCB_blank2A_20110509.raw (second blank in the run order after study A sample) PCB_B1_20110509.raw (study B, this is your first digestion, B1) PCB_blank1B1_20110509.raw (first blank in the run order after study B sample) PCB_blank2B1_20110509.raw (second blank in the run order after study B sample) 2nd week PCB_A_20110516.raw PCB_blank1A_20110516.raw PCB_blank2A_20110516.raw … … 5th week PCB_A_20110606.raw PCB_blank1A_20110606.raw PCB_blank2A_20110606.raw PCB_B2_20110606.raw (this is your second digestion, B2) PCB_blank1B2_20110606.raw PCB_blank2B2_20110606.raw …
Rule 4. Occurrence report Achieving long-term reproducibility is a daunting task for any proteomics core facility.  Our study also aims to identify components of LC-MS platforms that play a detrimental role in reproducibility and repeatability. To succeed in this task, it is important that participants report any relevant occurrence to their LC-MS system so as we can correlate such occurrences with sample profile changes over time. Some of the important occurrences that participants should reported are listed below: Column change MS maintenance LC maintenance S-lens cleaning Different trypsin batch To report any occurrence, you may write it on a .txt file and name it similar to your .raw files. For example, if you would like to report that a new column has been installed on July, 9th 2011, then you name the file PCB_20110709.txt and upload it in our ftp along with your raw files.
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