The simplest and most accurately characterized QC materials are mixtures of synthetic peptides, including those that have been isotopically labeled to avoid interference with peptides from the samples (Table ). Isotopically labeled or unlabeled, these peptide mixes fall under the QC1 category. The Pierce Peptide Retention Time Calibration Mixture, Biognosys iRT Kit, and Sigma-Aldrich MSRT1 standard are good examples, providing a mixture of unlabeled nonendogenous peptides that elute over a reverse phase gradient. These can be used to monitor chromatographic performance during the run, but also post-acquisition retention time normalization (or calibration into universal retention time scale). Mixtures of differentially isotopically labeled peptides are useful for checking quantitative accuracy and detection limits, which include the Pierce LC-MS/MS System Suitability Standard (7 x 5 mixture), Promega 6 × 5 LC-MS/MS Peptide Reference Mix, and the Sigma-Aldrich MSQC1 calibration standard for multiple-reaction monitoring (MRM) analyses. Due to the relative simplicity of these mixtures, they are normally spiked into a sample background, or, in the case of retention time standards, into every sample. They can be spiked into digested proteome QC materials (QC2) to create a QC3 (described below). These peptide mixtures provide very specific QC information which can easily be extracted in automated data analyses, and often have associated QC software (described below). Depending on when in the process the peptides are added, they inform more or less of the sample analysis. For instance, if the peptides are spiked into a sample following digestion but before peptide cleanup, the spike levels can be used to evaluate losses in the cleanup, which is critically important in samples with low protein concentration or single-cell analyses . Spiking into the sample earlier covers more steps of the experiment but increases the ambiguity in the QC results when something goes wrong and may necessitate additional analyses during troubleshooting. Peptides should be spiked in as early as necessary and as late as possible to bracket experimental steps.