The following Guidelines (below) and Checklist are provided to aid authors in preparing their manuscripts and to inform them of the items that must be included in their manuscripts.

Authors will need to complete the Checklist during the submission process to help ensure that their manuscripts contain all the required information. Authors are to note the page numbers where the information can be found in the paper. Manuscripts will be checked to ensure they comply with the guidelines at the same time that the peer review is being performed. It is important to note that as with all other papers in MCP, the compliance check does not constitute a review of the manuscript as this ONLY determines if the article conforms to the guidelines, i.e., contains the requisite information. The compliance check does not judge the quality of the data or evaluate the scientific suitability of the manuscript.

Guidelines for Targeted MS Manuscripts

Reporting the Tier Level of the Analyses Used: Targeted proteomic analyses can be divided into three strata differing by the intended application and extent of analytical validation. This Tier structure was developed as a direct outcome of a meeting sponsored by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) Program (see Carr et al. Mol Cell Proteomics 2014; PMID: 24443746). Tier 1 measurements have the highest standards and generally correspond to clinical bioanalyses / diagnostic laboratory tests that measure a single analyte (or small numbers of analytes). Tier 2 measurements are for research use assays, often highly multiplexed, for quantifying proteins, peptides and post-translationally-modified peptides. Tier 1 and 2 have two properties that together differentiate them from discovery experiments: 1) the ability to measure repeatedly sets of analytes of interest within and across samples/experiments and 2) they both employ internal standards for each analyte, for confident detection and precise quantification. Tier 3 measurements, useful in early-stage biological studies, enable repeatable measurement of the same sets of analytes across experiments but do not employ internal standards for either accurate or precise measurement of the levels of each analyte. Tier 3 methods do not constitute assays, but instead employ targeted strategies to bias towards detection of a predefined set of analytes. The manuscript requirements are dependent on the Tier level of analysis performed. In the cover letter and in the manuscript, authors should state the Tier level of the analyses they have developed and applied. Data-Independent Acquistion (DIA) studies are generally not targeted in their acquisition but are in their analysis. They have their own set of guidelines with which they have to comply.

For all Tiers, authors must provide the following written information:

• for human samples other than established cell lines, a Clinical Compliance check is required that specifically addresses how biofluids or tissue specimens were collected and handled, including IRB approval). Please see this page for details.
• name(s) of database(s) (e.g., PRIDE, MassIVE, etc.) where the raw data has been deposited and the access code(s) (see peptide/protein guidelines for information on raw data deposition).
• details of the experimental design and rationale, including statistical rationale for the design and/or regulatory guidance that was followed, as described in Section III of the Checklist, below.
• a subsection in the Experimental Methods with the header “Development and Analytical Validation Targeted MS Assays/Measurements” that includes the information described in section IV of the Checklist.
• the methods used for data collection and quantification for the samples and the values obtained including measurements of precision and/or accuracy.

For Tiers 1 and 2 analyses, authors are also required to provide all chromatograms used in quantification of analytes. This requirement (details for which are bulleted, below) is easily fulfilled using Skyline and Panorama (and possibly other tools in the future), and this tool combination also simplifies addressing the other requirements bulleted, below. If Skyline/Panorama or equivalent has not been used, authors may provide the above required chromatogram information in any format with a freely available viewer, such as PDF, as supplement at the time of first submission. Failure to provide either the necessary link to the Panorama files or equivalent, or to provide the chromatograms as pdf supplemental data may result in papers being returned without review.

As noted, this requirement pertains to Tiers 1 and 2 targeted analyses, only, and does not currently apply to label-free methods of quantification of the analytes (including cases where one or more labeled peptides have been used for normalization/quantification - but not for all analytes). These approaches fall under Tier 3.

• The chromatograms supplied must also include clearly marked integration boundaries or peak area shading used in the calculation of area under the curve (AUC), which was used as the basis for all reported quantities. Any smoothing or interpolation applied to the data must be reported and its relationship to the AUC calculation explained. The method for AUC calculation must be depicted visually using the graphed chromatograms and integration boundaries or peak area shading. If integration boundaries have been manually edited, this must be noted and details provided. A detailed explanation of the tool used to calculate the AUC from the graphed data must be provided. It is not required that the raw chromatogram point values graphed be made available. The actual (median) or estimated number of points across the peak should be reported for all the analytes in the assay (we recommend no fewer than 10-12 points across each peak). In the cases where actual value is not easily reported, an estimate may be provided from the cycle time or dwell time, retention time window used and number of concurrent transitions.
• If screen shots are provided (rather than the preferred mode of making data viewable with software such as Panorama), the chromatograms provided must include the full extent of the integrated peak, along with at least two peak widths of elution time (1 on either side) outside the peak itself to allow for some understanding of surrounding noise or potential interference. Chromatograms of both the analyte and isotopically-labeled standard (heavy peptide) must appear in a single view/page with matching retention time scales to allow reviewers and readers to ascertain coelution and assess shape matching.

At present, the simplest way to provide the required chromatogram information is to have used Skyline for method development and data analysis. The Skyline files can then be uploaded into Panorama Public into a restricted area for confidential reviewer-only access. A tutorial on use of Panorama Public can be found at PanoramaWeb.org. Regardless of what tools are used, authors should state the versions of each software tool that was used. MCP will update these guidelines as new tools become available that facilitate meeting the above requirements.